Transposon

ABSTRACT

A transposon which comprises an RNA polymerase recognition site and a homing endonuclease recognition site. The transposon may be used in methods for the identification of an essential gene or a conditional essential gene of an organism. Genes identified in such methods are useful as substrates for use in screening for antibacterials, antiparasitics, fungicides, pesticides and herbicides.

FIELD OF THE INVENTION

This invention relates to a new type of transposon. The transposon maybe used in methods for the identification of essential and conditionalessential genes, in particular in bacteria.

BACKGROUND OF THE INVENTION

The increase in prevalence of antibiotic-resistant bacteria, forexample, has renewed interest in the search for new targets forantibacterial agents. Essential genes and in particular the proteinswhich they encode may be good substrates for use in screens forantibacterials, antiparasitics, fungicides, pesticides and herbicides.Essential genes and their protein products potentially represent suchtargets.

Additionally, there is an interest in the identification of conditionalessential genes, that is genes which are essential for the survival ofan organism in a particular environment. In the case of pathogenicbacteria, for example, conditional essential genes include those whichare required for survival in a host. Such genes and the proteins whichthey encode may also be good targets for use in screens forantibacterials. In particular, bacteria which carry mutations in suchgenes may be useful in attenuated live vaccines.

SUMMARY OF THE INVENTION

Essential genes are those genes which, when missing (eg. because of achromosomal deletion) or mutated to render them non-functional, resultin a lethal phenotype. That is, they are genes without which an organismcannot survive. Conditional essential genes are those genes which,although not absolutely essential for the survival of an organism underall conditions, are essential for survival under various conditionalrestraints. Examples of particular conditional restraints includesurvival at elevated temperatures and survival of a pathogen within itshost.

A number of transposon-based strategies have been developed for theidentification of essential and conditional essential genes. We have nowdevised a new set of transposons. The transposons can be used in avariety of methods for the identification of essential and conditionalessential gene in the genome of an organism.

According to the invention there is thus provided a transposon whichcomprises an RNA polymerase recognition site and a homing endonucleaserecognition site.

The invention also provides:

use of a transposon of the invention in a method for the identificationof an essential or a conditional essential gene;

a method for identifying an essential gene of an organism, which methodcomprises:

(i) providing a library of transposon insertion mutants of the saidorganism, wherein the transposon is a transposon of the invention;

(ii) isolating chromosomal DNA from the library of (i);

(iii) digesting the chromosomal DNA with a restriction endonuclease thatis capable of cutting 5′ to the RNA polymerase recognition site(s) inthe transposon and 3′ to the RNA polymerase recognition site(s) in thechromosomal DNA flanking the transposon insertion site;

(iv) transcribing the resulting digested DNA from the RNA polymeraserecognition site(s) in the said DNA;

(v) hybridising the resulting RNA with an oligonucleotide array; and

(vi) identifying a probe on the oligonucleotide array which correspondsto an essential gene of the organism;

a method for identifying a conditional essential gene of an organism,which method comprises:

(a) providing a first sample of a library of transposon insertionmutants of the said organism (input library);

(b) providing a second sample of the library and subjecting that sampleto a conditional restraint;

(c) collecting the mutants that survive the conditional restraint instep (ii) to give a second library (output library);

(d) carrying out a method according to steps (ii) to (iv) of the methodset out above on the input library from step (a) and on the outputlibrary from step (c);

(e) hybridising the transcribed RNA derived from the input library andfrom the output library separately to copies of the same oligonucleotidearray or, if the RNA derived from the two libraries is differentiallylabelled, to the same oligonucleotide array; and

(f) identifying a probe on the oligonucleotide array(s) whichcorresponds to a conditional essential gene of the organism;

a method for identifying an essential gene of an organism, which methodcomprises:

(i) providing a library of transposon insertion mutants of the saidorganism, wherein the transposon is a transposon of the invention;

(ii) isolating chromosomal DNA from the library of (i);

(iii) digesting the chromosomal DNA with a restriction endonuclease thatis capable of cutting 5′ to the RNA polymerase recognition site(s) inthe transposon and 3′ to the RNA polymerase recognition site(s) in thechromosomal DNA flanking the transposon insertion site;

(iv) transcribing the resulting digested DNA from the RNA polymeraserecognition site(s) in the said DNA;

(v)′ reverse transcribing the resulting RNA;

(v)″ hybridising the resulting cDNA with an oligonucleotide array; and

(vi) identifying a probe on the oligonucleotide array which correspondsto an essential gene of the organism;

a method for identifying a conditional essential gene of an organism,which method comprises:

(a) providing a first sample of a library of transposon insertionmutants of the said organism (input library);

(b) providing a second sample of the library and subjecting that sampleto a conditional restraint;

(c) collecting the mutants that survive the conditional restraint instep (ii) to give a second library (output library);

(d) carrying out a method according to steps (ii) to (v)′ of the methodset out above on the input library from step (a) and on the outputlibrary from step (c);

(e) hybridising the reverse transcribed cDNA derived from the inputlibrary and from the output library separately to copies of the sameoligonucleotide array or, if the cDNA derived from the two libraries isdifferentially labelled, to the same oligonucleotide array; and

(f) identifying a probe on the oligonucleotide array(s) whichcorresponds to a conditional essential gene of the organism;

use of an essential or conditional essential gene identified by a methodas set out above, or a polypeptide encoded by a said gene, in a methodfor identifying an inhibitor of transcription and/or translation of thatgene and/or activity of a polypeptide encoded that gene;

a method for identifying an inhibitor of transcription and/ortranslation of an essential or conditional essential gene and/or aninhibitor of activity of a polypeptide encoded by a said gene, whichmethod comprises:

(a) identifying an essential or conditional essential gene by a methodas set out above; and

(b) determining whether a test substance can inhibit transcriptionand/or translation of a gene identified in step (a) and/or activity of apolypeptide encoded by a said identified gene, thereby to identify asaid inhibitor;

an inhibitor identified by such a method according to claim;

an inhibitor of the invention for use in a method of treatment of abacterial, fungal or eukaryotic parasite infection, wherein theessential or conditional essential gene used to identify the inhibitoris a bacterial, fungal or eukaryotic parasite essential or conditionalessential gene;

use of such an inhibitor in the manufacture of a medicament for use inthe treatment of a bacterial, fungal or eukaryotic parasite infection;

a pharmaceutical composition comprising such an inhibitor and apharmaceutically acceptable carrier or diluent;

a method of treating a host suffering from a bacterial, fungal oreukaryotic parasite infection, which method comprises the step ofadministering to the host a therapeutically effective amount of such aninhibitor;

a method for the preparation of a pharmaceutical composition, whichmethod comprises:

(a) identifying an inhibitor of transcription and/or translation of anessential or conditional essential gene of an organism and/or aninhibitor of activity of a polypeptide encoded by a said gene by amethod as set out above wherein the essential or conditional essentialgene is a bacterial, fungal or eukaryotic parasite essential orconditional essential gene; and

(b) formulating the inhibitor thus identified with a pharmaceuticallyacceptable carrier or diluent;

a method for treating a host suffering from a bacterial, fungal oreukaryotic parasite infection, which method comprises:

(a) identifying an inhibitor of transcription and/or translation of anessential or conditional essential gene of an organism and/or aninhibitor of activity of a polypeptide encoded by a said gene by amethod as set out above wherein the essential or conditional essentialgene is a bacterial, fungal or eukaryotic parasite essential orconditional essential gene;

(b) formulating the inhibitor thus identified with a pharmaceuticallyacceptable carrier or diluent; and

(c) administering to the host a therapeutically effective amount of aninhibitor thus formulated;

an inhibitor of the invention, wherein the essential or conditionalessential gene is a plant bacterial, plant fungal or plant pestessential or conditional essential gene;

use of such an inhibitor as a plant bactericide, fungicide or pesticide;

an inhibitor of the invention, wherein essential or conditionalessential gene is a plant essential or conditional essential gene;

use of such an inhibitor as a herbicide;

a bacterium attenuated by a non-reverting mutation in one or more genesidentified by a method of the invention;

a vaccine comprising such a bacterium and a pharmaceutically acceptablecarrier or diluent;

a bacterium as described above for use in a method of vaccinating ahuman or animal;

use of such a bacterium for the manufacture of a medicament forvaccinating a human or animal;

a method for raising an immune response in a mammalian host, whichmethod comprises the step of administering to the host a bacterium asset out above;

a method for preparing an attenuated bacterium, which method comprises:

(a) identifying a conditional essential gene in a bacterium by a methodof the invention; and

(b) introducing a non-reverting mutation into a thus-identifiedconditional essential gene of the bacterium, thereby to attenuate thebacterium;

a method for the preparation of a vaccine, which method comprises:

(a) identifying a conditional essential gene in a bacterium by a methodof the invention;

(b) introducing a non-reverting mutation into a thus-identifiedconditional essential gene of the bacterium, thereby to attenuate thebacterium; and

(c) formulating the attenuated bacterium with a pharmaceuticallyacceptable carrier or diluent; and

a method for raising an immune response in a mammalian host, whichmethod comprises:

(a) identifying a conditional essential gene in a bacterium by a methodof the invention;

(b) introducing a non-reverting mutation into a thus-identifiedconditional essential gene of the bacterium, thereby to attenuate thebacterium;

(c) formulating the attenuated bacterium with a pharmaceuticallyacceptable carrier or diluent; and

(d) administering to the host the attenuated bacterium thus formulated.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a diagrammatic representation of the “Gene Kelly”transposon consisting of ME (mosaic end IS sequences), binding sites forthe SP6 and T7 RNA polymerases, homing endonuclease sites for I-SceI andPI-PspI, an R6k origin of replication and a kanamycin resistancecassette.

FIG. 2 sets out the sequence of the “Gene Kelly” transposon.

FIG. 3 shows a diagrammatic representation of the original epicentreEZ:Tn R6k ori Kan transposon with the oligonucleotide sequencesoverlaid. Black boxes represent matching sequence between PCRoligonucleotides and the epicentre transposon.

FIG. 4 sets out a graph showing the position of insertion in the LT2genome of the 46 sequenced transposon mutants (position in base pairs inincreasing number order against number of transposons). From the graphit can be seen that there is a fairly random distribution of insertionsthroughout the genome.

FIG. 5 sets out a diagram of each end of the transposon showing therelative position of the iPCR oligonucleotides with the restrictionendonuclease cut sites and RNA polymerase promoters in transposedchromosomal DNA, which has been digested and religated.

FIG. 6 sets out a schematic diagram of the ligation capture method ofrecovering the ends of the transposon.

FIG. 7 sets out array hybridisation results comparing input and outputpool data. Array Images, panel a, reveals comparable sections of thescanned microarrays resulting from the hybridisation of the labeled RNAtarget generated from the input and output pool restricted DNA. Sets ofprobes corresponding to three transposon mutants (a set of probes refersto probes synthesised in both the sense and anti-sense directions aroundthe point of transposon insertion. These are shown as horizontal linesabove, sense probes, and below, anti-sense probes, the disrupted locus),1 (corresponding to probes encompassing a transposon insertion withinthe aroA gene), 2 (corresponding to probes encompassing a transposoninsertion within gene X) and 3 (corresponding to probes encompassing atransposon insertion within an intergenic region) are boxed in the inputpanel and the data extracted from the input and output arrays arecompared in the adjacent panel b, Extracted Data. In panel b, dashedvertical lines shown between the sense and anti-sense probes refer tothe position of RsaI restriction endonuclease recognition sequences.Boxes above and below the black line indicate genes either in the senseor anti-sense direction, respectively, relative to the published LT2genome sequence.

FIG. 8 sets out insertion site relative to S. aureus MW2 genome sequenceof 59 Tn917, 50 Tn551 and 86 Mariner Erm Gene Kelly mutants generated inS. aureus SH1000.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 sets out the sequence of the “Gene Kelly” transposon.

SEQ ID NO: 2 sets out the sequence of primer 97, which was used in theconstruction of the “Gene Kelly” transposon.

SEQ ID NO: 3 sets out the sequence of primer 98, which was used in theconstruction of the “Gene Kelly” transposon.

SEQ ID NO: 4 sets out the sequence of primer 107, which can be used tocarry out iPCR in a protocol for generating RNA run-offs.

SEQ ID NO: 5 sets out the sequence of primer 115, which can be used tocarry out iPCR in a protocol for generating RNA run-offs.

SEQ ID NO: 6 sets out the sequence of primer 116, which can be used tocarry out iPCR in a protocol for generating RNA run-offs.

SEQ ID NO: 7 sets out the sequence of primer 108, which can be used tocarry out iPCR in a protocol for generating RNA run-offs.

SEQ ID NO: 8 sets out the sequence of primer 117, which can be used tocarry out iPCR in a protocol for generating RNA run-offs.

SEQ ID NO: 9 sets out the sequence of primer 118, which can be used tocarry out iPCR in a protocol for generating RNA run-offs.

SEQ ID NO: 10 sets out the sequence of primer 113, which can be used ina protocol for ligation capture recovery of “Gene Kelly” transposonends.

SEQ ID NO: 11 sets out the sequence of primer 114, which can be used ina protocol for ligation capture recovery of “Gene Kelly” transposonends.

SEQ ID NO: 12 sets out the sequence of primer 135, which was used in theconstruction of the “Mariner Erm Gene Kelly” transposon.

SEQ ID NO: 13 sets out the sequence of primer 136, which was used in theconstruction of the “Mariner Erm Gene Kelly” transposon.

SEQ ID NO: 14 sets out the sequence of primer 5′ erm, which can be usedin the isolation of an erythromycin resistance marker gene sequence.

SEQ ID NO: 15 sets out the sequence of primer 3′ erm, which can be usedin the isolation of an erythromycin resistance marker gene sequence.

SEQ ID NO: 16 sets out the sequence of primer 12, which can be used tosequence the “Mariner Erm Gene Kelly” transposon.

SEQ ID NO: 17 sets out the sequence of primer 13, which can be used tosequence the “Mariner Erm Gene Kelly” transposon.

SEQ ID NO: 18 sets out the sequence of primer 199, which was used tosequence chromosomal DNA from S. aureus which flanked Mariner Erm GeneKelly transposon insertion sites.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a new transposon which is suitable for use inmethods for the identification of essential and conditional essentialgenes. The transposon has been named the “Gene Kelly” transposon.

The transposon of the invention is typically a modified Tn5 or Marinertransposon, although, in principle, any transposon may be modified so asto prepare a transposon according to the invention. The transposon ofthe invention has a combination of features which make it a versatiletool for use in a number of protocols for the identification ofessential and conditional essential genes. We refer to these methods astransposon mediated differential hybridisation (TMDH) techniques.

A transposon of the invention comprises an RNA polymerase recognitionsite (sometimes referred to as an RNA polymerase recognition sequence)and a honing endonuclease recognition site (sometimes referred to as ahoming endonuclease recognition sequence).

Typically, the RNA polymerase recognition site is located proximal to anend of the transposon, for example adjacent to mosaic ends (if thetransposon has them). The RNA polymerase recognition site is typicallyoriented so that it directs transcription out of the transposon itself.Thus, DNA sequence flanking the integration site of the transposon maybe transcribed.

These transcribed sequence originate from DNA sequence flanking atransposon insertion site. Such DNA sequences are therefore susceptibleto insertion and are unlikely to represent essential gene sequences.

Sequences flanking a transposon insertion site may therefore be isolatedfrom a library of transposon insertion mutants (generated using atransposon of the invention) and hybridised with an oligonucleotidearray which comprises probes corresponding to open reading frames froman organism to be studied. If the library of insertion mutants comprisesa transposon insertion in all of the non-essential genes of theorganism, any probe in the oligonucleotide array to which none of theflanking sequences hybridise is likely to be a good candidate fororiginating from an essential gene. Typically, oligonucleotide arrayssuitable for use with a transposon comprise probes corresponding to allof the open reading frames from the organism in question and thereforepotentially all of the essential genes of an organism may be identifiedsimultaneously.

Typically, a transposon of the invention comprises two RNA polymeraserecognition sites located proximal to the ends, typically to theopposite ends, of the transposon. Preferably, both RNA polymeraserecognition sites point out of the transposon, i.e. are capable ofdirecting transcription or DNA sequence flanking the transposoninsertion site.

Preferred transposons of the invention comprise two diverse (different)RNA polymerase recognition sites, although the two sites may be thesame. The use of two RNA polymerase recognition sites allows twoseparate pools of RNAs to be isolated from a library of Gene Kellyinsertion mutants. One pool will correspond to DNA sequences flankingone side of the transposon insertion site and the other pool willrepresent sequences flanking the other side of the transposon insertionsite (these may be referred to as right- and left-hand pools). Thegeneration of these two separate pools may help to minimise the risk ofan essential gene being incorrectly assigned as a non-essential gene.This is explained in more detail below.

A transposon of the invention also comprises a homing endonucleaserecognition site. Homing endonucleases are rare cutters, especially inbacterial DNA. Incorporation of recognition sites for such endonucleasesinto a transposon effectively permits the introduction of these sitesinto the genome being studied. Transposed DNA may be digested with theappropriate homing endonuclease and the resulting ends (if none arepresent in the bacterial genome) should therefore all originate from theGene Kelly transposon. The fragments resulting from digestion with ahoming endonuclease may then be digested with a restriction endonucleasewhich cuts in the genome of organism being studied. The resultingtransposon:flanking sequence fragments may be rescued using aligation-capture technique described below, allowing the rapid andselective purification of regions of the genomic DNA of the organismbeing studied which originate from a site of transposon insertion. Suchregions can be used in hybridisation experiments with oligonucleotidearrays as outlined above and as described in more detail below.

Preferably, if the transposon comprises two RNA polymerase recognitionsites, it will also comprise two homing endonuclease recognition sites.Typically, if two homing endonuclease recognition sites are present,they will be diverse (different) homing endonuclease recognition sites.However, they may be the same endonuclease recognition site. The use oftwo, typically diverse, homing endonuclease recognition sites incombination with two, typically diverse, RNA polymerase recognitionsites may allow two separate pools of sequences flanking either side ofthe inserted transposons to be isolated separately i.e. a left-hand pooland a right-hand pool of flanking sequences may be separated. Thegeneration of these two pools may help to minimise the risk of anessential gene being incorrectly assigned as a non-essential gene. Thisis explained in more detail below.

In addition, a transposon of the invention may incorporate a bacterialorigin of replication. This allows plasmid-rescue of the completetransposon to be carried out (plus the flanking regions of chromosomalDNA around the insertion site). This may be achieved by digestion ofgenomic DNA isolated from a library of transposon insertion mutants(with a restriction endonuclease that does not cut in the transposonsequence or at least in the bacterial origin of replication in thetransposon sequence), religation and then transformation into a strainof bacteria in which the origin of replication will function. A suitablebacterial origin of replication is the R6k origin of replication.

A transposon of the invention thus comprises two critical features: (i)an RNA polymerase recognition site; and (ii) a homing endonucleaserecognition site. Optionally, a bacterial origin of replication may bepresent. A more preferred version of the Gene Kelly transposon comprisestwo diverse RNA recognition sites and two diverse homing endonucleaserecognition sites. The full sequence of a preferred Gene Kellytransposon is set out in FIG. 2 and SEQ ID NO: 1.

Transposons, sometimes called transposable elements, are mobilepolynucleotides. The term transposon is well known to those skilled inthe art and includes classes of transposons that can be distinguished onthe basis of sequence organisation, for example short inverted repeatsat each end; directly repeated long terminal repeats (LTRs) at the ends;and polyA at 3′ ends of RNA transcripts with 5′ ends often truncated.Some types of virus also integrate into the host genome, for exampleretroviruses, and may therefore be used to generate libraries ofinsertion mutants. However, transposons are typically preferred toviruses because issues of safety related to pathogenicity may beavoided.

A transposon of the invention comprises an RNA polymerase recognitionsite (typically two) and a homing endonuclease recognition site(typically two). Any suitable transposon may be modified to produce atransposon of the invention. Suitable transposons for use in bacteriawhich can be modified to generate a Gene Kelly transposon include Tn3,γδ, Tn10, Tn5, TnphoA, Tn903, Tn917, Mariner Bacteriophage Mu andrelated viruses. Any of the above mentioned transposons may be modifiedto generate a transposon of the invention. Different parts of differenttransposons may be mixed and matched in a transposon of the invention. Aparticular preferred transposon for use in the invention is a modifiedTn5 transposon.

Suitable transposons for use in fungi which can be modified to generatea transposon of the invention include the Ty1 element of Saccharomycescerevisiae, the filamentous fungi elements (the filamentous fungiinclude agriculturally important plant pathogens such as Erysiphegraminis, Magnaporthe grisea) such as Fot1/Pogo-like andTc1/Mariner-like elements (see Kempen and Kuck, 1998, Bioessays 20,652-659 for a review of such elements).

Suitable transposons for use in plants which can be modified to generatea transposon of the invention include Ac/Ds, Tam3 and other Tamelements, cin4 and spm.

Suitable transposons for use in animals which can be modified togenerate a transposon of the invention include P and hobo which may beused in Drosophila and Tc1 which can be used in Caenorhabditis elegans.

A preferred transposon of the invention is one which carries anantibiotic resistance gene (which may be useful in identifying mutantswhich carry a transposon) conferring resistance to, for example,kanamycin (in particular for use with a Tn5 -based transposon),erythromycin (in particular for use with a Mariner-based transposon)streptomycin or bleomycin.

A transposon of the invention may comprise one, two or more recognitionsites for an RNA polymerase. Preferred recognition sites are those forwhich the corresponding RNA polymerase is highly selective forinitiation. Other preferred recognition sites are those for which thecorresponding RNA polymerase does not initiate transcription fromsequences of the organism being studied. Preferred examples of RNArecognition sites are those recognised by bacteriophage RNA polymerases,for example the recognition site for T7 RNA polymerase, T3 RNApolymerase or SP6 RNA polymerase, in particular the T7 RNA polymeraserecognition site. A preferred. Gene Kelly transposon will thus carry twoof a 17 RNA polymerase recognition site, an SP6 RNA polymeraserecognition site or a T3 RNA polymerase recognition site, for example aT7 RNA polymerase recognition site and an SP6 RNA polymerase recognitionsite. The recognition sites for these specific RNA polymerases are wellknown to those skilled in the art

The RNA polymerase recognition site may appear anywhere within atransposon for use in the invention. However, typically the RNApolymerase recognition site will be located proximal to one end of thetransposon, i.e. proximal to one IS/ME. Typically, the 3′ end of the RNApolymerase recognition site will be situated from one to 30, for examplefrom five to twenty base pairs away from the 5′ end of one of the IS/ME.

Preferred transposons of the invention comprise two RNA polymeraserecognition sites, which generally will be different RNA polymeraserecognition sites. For example, a transposon may comprise a T7 RNApolymerase recognition site and an SP6 RNA polymerase recognition site.

A transposon of the invention may also comprise more than two RNApolymerase recognition sites, for example three or four RNA recognitionsites. More than two recognition sites may be useful in a situationwhere the genome of an organism being studied possesses recognitionsites for one of the RNA polymerase recognition sites present in thetransposon. Thus, different RNA polymerase sites in one transposon maybe suitable for use in different genomes. The specific RNA polymeraserecognition sites used may vary with the particular organism beingstudied. A single transposon may therefore be suitable for use in anumber of different organisms.

A transposon of the invention comprises a homing endonucleaserecognition site. Preferably, two such sites are present which may bethe same or diverse. Homing endonucleases are also known as intron orintein encoded endonuclease. They are encoded by genes with mobile,self-splicing introns or inteins (protein introns). Any homing nucleaserecognition site may be used, for example I-SceI, PI-PspI or I-PpoI, orpreferably a combination of any two thereof.

A homing endonuclease recognition site may be located anywhere in thetransposon, but typically it is situated 5′ to an RNA polymeraserecognition site, i.e. further into the transposon than the RNApolymerase recognition site. Preferred transposons of the invention maycomprise two homing endonuclease recognition sites which generally willbe different homing endonuclease recognition sites, for example a I-SceIand a PI-PspI recognition site. This is usually the case where thetransposon comprises two RNA polymerase recognition sites.

A transposon of the invention may, however, comprise more than twohoming endonuclease recognition sites, for example three or four homingendonuclease recognition sites. More than two recognition sites may beuseful in a situation where the genome of an organism being studiedpossesses recognition sites for one of the homing endonucleaserecognition sites present in the transposon. Thus, different homingendonuclease recognition sites in one transposon may be suitable for usein different genomes. The specific homing endonuclease recognition sitesused in the transposon may vary with the particular organism beingstudied. A single transposon may therefore be suitable for use in anumber of different organisms.

Typically, a transposon of the invention will comprise the same numberof RNA polymerase recognition sites as homing endonuclease recognitionsites, ideally two of each. Each homing endonuclease recognition sitewill typically be located 5′ to an RNA polymerase recognition site, forexample such that the 3′ end of the homing endonuclease recognition siteis from one to 30, for example, from five to twenty base pairs away fromthe 5′ end of one of an RNA polymerase recognition site.

A transposon of the invention may also comprise a bacterial origin ofreplication. A preferred example of a suitable bacterial origin ofreplication is the R6k origin of replication. The R6k origin ofreplication is capable of functioning in a pir⁺ strain of bacteria.

A transposon of the invention may be used in methods for theidentification of essential or conditional essential genes, i.e. use ofa transposon of the invention in a method for the identification of anessential gene or a conditional essential gene is provided according tothe invention. The methods described below for the identification ofessential or conditional essential genes using a transposon of theinvention can conveniently be referred to as transposon mediateddifferential hybridisation (TMDH). TMDH techniques are described indetail in WO-A-01/07651 (PCT/GB00/02879) and the transposon of theinvention may be used in any of the methods set out therein.

In methods for the identification of essential gene, typically the firststep is the provision of a library of transposon insertion mutants.Libraries of insertion mutants using a transposon of the invention maybe generated according to any method known to those skilled in the art.For example, libraries of bacterial transposon insertion mutants can beconstructed using either plasmid or bacteriophage vectors containing thetransposon and a selectable marker. Bacteriophage λ, eg. λTnphoA can beused to infect a suitable recipient bacterial strain, for example E.coli XAC. This E. coli strain has a suppressor mutation which preventsthe bacteriophage from replicating and subsequently lysing and alsocontains an antibiotic resistance gene to allow selection of coloniescontaining transposed chromosomal DNA. The vector contains mutation(s)preventing integration of the λ chromosome into the bacterial hostchromosome and thus the growth of false positive colonies without amutated E. coli gene is prevented. Cultures of the recipient strain aregrown in enriched medium (eg. Luria Broth) and cells in mid log phase ofgrowth are infected with the λ transposon vector for 1 hour at 37° C.Aliquots of the infected cells are plated out on L-agar supplementedwith the appropriate selective antibiotic and grown overnight at 37° C.These colonies constitute a transposon library and can be furtheranalysed by the TMDH procedure described in this application.

Alternatively, transposome complexes comprising the transposon in acomplex with a transposase may be generated and electroporated into asuitable electrocompetent host. Suitable techniques for preparingtransposomes and for electroporating transposomes into host cells arewell known to those skilled in the art.

Growth of such libraries results in the generation of potentiallythousands of insertion mutants all of which mutants carry insertion thatare, of necessity, in genes that (when mutated) do not result in thedeath of the cell ie. are non-essential genes.

Each mutant in a suitable transposon insertion library may carry onetransposon insertion. However, a mutant may carry more than onetransposon insertion, for example two, three, four, five, ten or twentytransposon insertions. A transposon insertion mutant library suitablefor use in the invention will comprise at least one transposon insertionmutant for at least 60%, at least 70%, typically at least 80%,preferably at least 90%, more preferably at least 95%, even morepreferably at least 99%, or most preferably substantially all of thenon-essential genes in the organism being studied. Preferably thelibrary will be a saturating library, i.e. the library comprises atransposon insertion mutant for substantially all genes of the organismthat when mutated give rise to viable organisms.

A transposon insertion could be in an open reading frame of a gene or ina regulatory sequence of gene.

Any non-essential gene in the transposon insertion library may berepresented by more than one insertion mutant, for example two, three,four, five or up to ten insertion mutants, each carrying transposoninsertions at the same or different sites in the non-essential gene orcarrying insertions at the same site in different orientations.Preferred libraries will have, on average, more than one differenttransposon insertion mutant for each non-essential gene represented inthe library, for example at least two on average, at least four onaverage, at least 5 on average or at least 10 on average differenttransposon insertion mutants for each non-essential gene represented inthe library.

Some regions of a particular genome may be inaccessible to insertion bya particular transposon, for example because of a particular secondaryor tertiary structure which is inaccessible to a particular transposon.Thus it may be advantageous to combine two transposon libraries, therebyincreasing the probability of obtaining transposon insertions in agreater number of genes. For example, in the case of bacteriallibraries, a library generated with Gene Kelly transposons based on aTn5 transposon and a library generated with a Gene Kelly transposonsbased on a Tn10 transposon could, for example, be combined.

Generally, flanking sequence will be isolated from at least 60%, forexample at least 70%, at least 80%, preferably at least 90%, morepreferably at least 95% and most preferably at least 99% of thetransposon insertion mutants in a particular library of mutants.

In the method of the invention chromosomal DNA is prepared from thelibrary of transposon insertion mutants. Techniques for the isolation ofchromosomal DNA, alternatively referred to as genomic DNA, are wellknown to those skilled in the art. The transposons of the inventionallow for a number of different techniques to be used for the generationof RNA target sequences from the isolated genomic DNA.

In one version of TMDH, the chromosomal DNA thus prepared is thendigested with a restriction endonuclease. The restriction endonucleaseis one which is capable of cutting at a recognition site which islocated in the transposon at a position 5′ to the RNA polymeraserecognition site (which is located in the transposon) and 3′ to the RNApolymerase recognition site in the chromosomal DNA flanking thetransposon insertion site.

In a modification of this protocol, two restriction endonucleases may beused: a first restriction enzyme which cuts 5′ to the RNA polymeraserecognition site (which is located in the transposon) in the transposonitself; and a second restriction enzyme which cuts 3′ to the RNApolymerase recognition site in the chromosomal DNA flanking thetransposon insertion site. If two restriction enzymes are used in thisway, they may be used simultaneously or one after the other in eitherorder.

The exact restriction enzyme(s) to be used will depend on the sequenceof the transposon. However, typically an restriction endonuclease isused which has recognition sites that appear frequently within thegenome of the organism being studied. Thus, a series of DNA fragments isgenerated, some of which comprise an RNA polymerase recognition sitefused to a portion of flanking sequence, i.e. non-essential genesequence.

Generally, suitable restriction endonucleases will have six base pair,five base pair or preferably four base pair recognition sequences.Suitable examples of four base pair cutters are set out in Table 1below: TABLE 1 Examples of 4 bp recognition type II restrictionendonucleases suitable for use in TMDH Enzyme Recognition Site AciIC¹CGC GGC₁G AluI AG¹CT TC₁GA BfaI C¹TAG GAT₁C BstuI CG¹CG GC₁GC DpnI¹GATC CTAG₁ HaeIII GG¹CC CC₁GG HinpI G¹CGC CGC₁G HhaI GCG¹C C₁GCG MseIT¹TAA AAT₁T MspI C¹CGG GGC₁C NlaIII ¹CATG GTAC₁ RsaI GT¹AC CA₁TG Sau3a¹GATC CTAG₁ TaqI T¹CGA AGC₁T Tsp509 ¹AATT TTAA₁

The resulting fragments comprise an RNA polymerase site adjacent tonon-essential gene sequence. The fragments may optionally be sizeselected. If size selection is carried out, fragments with a size offrom about 100 bp to about 2000 bp or preferably of from about 200 bp toabout 600 bp may be isolated, for example from a gel, and purified. Thesmaller the fragments isolated, the smaller the chance of the RNA targetsequences including sequences from genes which lie next to genes whichhave been interrupted by transposons. If such adjacent sequences werefrom essential genes, there is the possibility that essential genesequences could be identified as non-essential gene sequences. Thus,size fractionation may reduce the amount of false non-essential genesequences.

RNA target sequences are generated from the DNA (host organism)sequences that flank the transposons, i.e. those regions correspondingto non-essential gene sequences. That is, the transposon:flankingsequence fragments are then used to generate the RNA sequences. Thus,following digestion, the transposon:flanking sequence fragments (the“target”) may be transcribed. Optionally, the transposon:flankingsequence fragments may be amplified prior to transcription, for exampleby PCR. Preferably this is carried out by iPCR (inverse PCR).

Transcription is carried out by in vitro transcription from the RNApolymerase recognition sequence. Techniques for carrying out in vitrotranscription are well known to those skilled in the art and anysuitable technique may be used. In essence, an RNA polymerase andribonucleotides are used.

The RNA target sequences so-generated may then be hybridised witholigonucleotide arrays. Alternatively, the RNA target sequences may bereverse transcribed to produce cDNA target sequences. Reversetranscription may be primed using an oligonucleotide having a sequencebased on the transposon sequence immediately 3′ to the site oftranscription initiation. Techniques for carrying out reversetranscription are well known to those skilled in the art and anysuitable technique may be used. In essence, a reverse transcriptase anddeoxyribonucleotides are used.

Preferably, the transcription reaction or the reverse transcriptionreaction is carried out in the presence of one or more labelledribonucleotides or one or more labelled deoxyribonucleotidesrespectively, so that the resulting RNA or cDNA target sequences arelabelled.

Suitable labels include radioactive labels, for example ³²P, ³³P or ³⁵S,or non-radioactive labels, for example an enzyme, a fluorescent label orbiotin.

Fluorescent labels are preferred, for example a water-solublefluorescent dye such as Cy3™ or Cy5™ or a fluorescein-tagged compoundsuch as FluorX™ (the NHS ester of carboxyfluorescein with an extendedlinker arm), fluorescein isothiocyanate (FITC) or5-([4,6-Dichlorotriazin-2-yl]amino)fluorescein (DTAF). Generally, itwill only be necessary to have one of the four ribonucleotides ordeoxyribonucleotides labelled.

The techniques described above allow the isolation of sequences flankingthe transposons in a library of transposon insertion mutants. Thus, apool of flanking sequences is generated collectively referred to as theRNA (or cDNA) target sequences. Although fragments in the pool aregenerated from only one side of the transposons, a transposon is capableof inserting at any particular locus (that can be disrupted) in eitherorientation. Thus, particularly in a saturating transposon insertionlibrary, many loci will be represented by mutants carrying insertions inboth orientations. Therefore, the RNA (or cDNA) target sequencesgenerated according to the TMDH method of the invention will, for manyloci, comprise flanking sequence in both orientations.

In a modification of this technique, the fragments comprising the RNApolymerase site fused to non-essential gene sequence may be amplified.

Amplification may be carried out by ligating linkers, preferablyvectorette units, to the fragments. If linkers are ligated to thefragments, the resulting fragments may be re-purified for examplethrough a gel or by using spun-column chromatography. PCR may thencarried out using the fragments as templates with a primer paircomprising an oligonucleotide specific for a transposon sequence and asecond oligonucleotide specific for a linker (eg. a vectorette)sequence. The use of transposon- and vectorette-specific PCR primersresults in the specific amplification of sequences that are adjacent tothe sites of transposon insertion.

Alternatively, the fragments may be amplified by cycle primer extension.The use of a suitable labelled oligonucleotide primer can allow theamplification of sequences adjacent to the sites of transposoninsertion. Those labelled amplified sequences can be used directly inhybridisation experiments.

Alternatively, the fragments may be amplified by inverse PCR (iPCR).Thus, the fragments may be self-ligated and subsequently amplified usingtransposon specific primers.

Suitable techniques for carrying out self-ligation are well known tothose skilled in the art. Any suitable ligase may be used, for exampleT4 DNA ligase. Ligation reactions may be carried out for from 6 to 24hours, for example from 12 to 16 hours at a temperature of from 10° C.to 20° C., for example at about 16° C.

Self-ligated molecules are then amplified using iPCR. Techniques forcarrying out iPCR are well known to those skilled in the art and may becarried out according to any suitable technique. Typically, iPCR iscarried out using two olignucleotides which bind divergently at alocation 5′ to the RNA polymerase recognition site. Preferably theoligonucleotides bind divergently to a location which is 3′ to therestriction endonuclease recognition site in the transposon. That is,the two olignucleotide recognition sites are preferably located on thetransposon between the restriction endonuclease recognition site and theRNA polymerase recognition site.

When using iPCR techniques, there is the possibility that, a “stuffer”fragment may ligate into the self-ligation reaction, which will beamplified along with the transposon-disrupted sequence. If this materialwere to be used in subsequent generation of the RNA target sequences,the stuffer sequence could create non-specific background signal as itwould also be hybridized to the high density array. In order to removethis stuffer fragment, the sequences amplified in iPCR can be redigestedwith whichever enzyme was used to isolate the transposon-flankingsequence fragments in the first place. This results in the release ofthe stuffer fragments which can be removed from the transposon:flankingsequence fragments. Removal of the stuffer fragments can be facilitatedif a biotinylated primer is used in iPCR The biotinylatedtransposon:flanking sequence fragments can then be removed from thestuffer fragments using a magnetic-bead-streptavidin conjugate.

Additional methods for amplifying transposon:flanking sequence fragmentsinclude, for example, splinkerette-PCR, targetted gene walking PCR,restriction site PCR, capture PCR, panhandle PCR and boomerang DNAamplification (for a review of these techniques see Hui et al., CellMol. Life Sci. 54 (1998) 1403-1411).

It is possible that the particular restricted endonuclease used will notcut within the gene in which the transposon is inserted, or cuts at alarge distance, for example more than 2 kb, away from the insertionsite. Therefore, if size selection is carried out, sequences from thesegenes may be lost. Thus, the generation of fragments may be carried outseveral times, each time using a different restriction endonuclease andthe resulting fragments may subsequently be pooled. The greater thenumber of enzymes used to make fragments, the greater the likelihood ofsequences from non-essential genes being represented in the final poolof fragments.

In a further modification of TMDH method, the chromosomal DNA isolatedfrom a transposon insertion library may be divided into a number ofaliquots. Those aliquots may then each be separately digested with adifferent restriction endonuclease which is capable of cutting at arecognition site which is located in the transposon at a position 5′ tothe RNA polymerase recognition site and in the chromosomal DNA flankingthe transposon 3′ to the RNA polymerase recognition site (which is inthe transposon). The chromosomal DNA may be separated into, for exampletwo, three, four, five or ten aliquots which are each separatelydigested with a different restriction endonuclease. Preferredrestriction enzymes are as set out in Table 1 above.

Thus, for example, two or three aliquots of the chromosomal DNA may beseparately digested with different suitable restriction endonucleases,for example two or three of HaeIII, HhaI, Hpych4IV and RsaI.

If the TMDH protocol is used in this modified format, the differentaliquots may be repooled after digestion and treated together in thesubsequent steps of TMDH. Alternatively, the digested aliquots may betreated separately in the subsequent steps. If this TMDH format isadopted, a number of pools of RNA target sequences result. Each pool ofRNA target sequences may be labelled with a different, for examplefluorescent, label.

In an alternative protocol for the preparation of RNA/cDNA targetsequences, the genomic DNA isolated from a library of transposon mutantsis digested with a homing endonuclease. Homing endonuclease recognitionsites are rare and therefore any “ends” generated by digestion with ahoming endonuclease should originate from the transposon. Fragmentsresulting from digestion with a homing endonuclease are then furtherdigested with a restriction endonuclease which does not cut in thetransposon, for example a restriction enzyme as described above, butwhich does cut within the genomic sequence flanking the transposon ends(i.e. flanking the transposon insertion site). The resultingtransposon:flanking sequence fragments may then be rescued by annealinga biotinylated linker to them and then isolating the biotinylatedtransposon:flanking sequence fragments with streptavidin-coatedparticles, for example streptavidin-coated magnetic beads. The linkeranneals to the homing endonuclease recognition site end of thefragments.

Alternatively the ends bearing the homing endonuclease recognition sitemay be rescued by labelling with digoxygenin and isolating the labelledfragments with an antibody raised against digoxygenin. The step ofdigestion with a restriction enzyme may be carried out after thetransposon:flanking sequence fragments have been isolated.

The use of homing endonucleases may allow the step of isolating genomicDNA from a library of transposon mutants to be eliminated. Thus, thelibrary of mutants may be digested directly with a homing endonuclease.Typically, an extract of the library may be generated. For example, ifthe library is a bacterial library, the baterial cells may be lysedbefore digestion with a homing endonuclease is carried out. Oncedigestion with a homing endonuclease has been carried out, transposonends (transposon:flanking sequence fragments) may be recovered asdescribed above.

The transposon:flanking sequence fragments are then used to generate theRNA target sequences by carrying out in vitro transcription from the RNApolymerase recognition site. In vitro transcription can be carried outas described above. If, after cutting with the homing endonuclease, afurther restriction enzyme is not used to cut within the flankingsequences, in vitro transcription may be carried out in the presence ofa dideoxyribonucleotide. That allows transcription to be terminated,reducing the risk of the transcribed sequence comprising portions ofgenes adjacent to the gene into which insertion has taken place.

The sequences which comprise the RNA target sequences (whichever of themethods described above is used to generate them) may be used forhybridisation with olignucleotide arrays. Oligonucleotide arrays used inthe TMDH protocol of the invention are preferably high informationcontent arrays.

Oligonucleotide arrays suitable for use in the invention may comprisesequences from one or more loci of a genome. Preferably suitableoligonucleotide arrays will represent at least 80% of all open readingframes (ORFs), more preferably at least 90% of all ORFs, for example 95%of all ORFs, even more preferably 99% of all ORFs or substantially allORFs of the genome represented on the oligonucleotide array.

By high information content array is meant an array in which there are ahigh number of probes covering the locus, loci or genome represented bythe array. For example, in a high information content array there may bea probe, for example, for every 30 to 500 base pairs of the locus, locior genome represented by the array. Preferably there will be a probe,for every 60 to 250 base pairs of locus, loci or genome represented inthe array, for example about every 100 base pairs. Probes may overlap,for example by 1, 2, 3, 4, 5, up to 10, up to 20, up to 30, up to 40 orup to 50 bases.

The olignucleotide probes on the array are, for example, from about 8 or9 to about 150 nucleotides in length, preferably from about 30 or 50 toabout 100 nucleotides in length or more preferably about 60 nucleotidesin length.

The oligonucleotide probes used in the array will typically be designedon the basis of the wild type sequence of the organism being studied.The oligonucleotide probes may be designed so that each probe hasminimal or substantially no cross-hybridisation with other sequences inthe genome from which the probes originate. The BLAST program can beused to design suitable probes (Altschul et al., J. Mol. Biol. 215.403-410).

Methods for making oligonucleotide arrays are well known to thoseskilled in the art.

Probes which show no hybridisation or substantially no hybridisation(there may be a low level of background non-specific hybridisation) withthe RNA target represent sequences that are unlikely to have beendisrupted by a transposon insertion event and consequently are strongcandidates for sequences corresponding to essential genes.

However, it is theoretically possible for oligonucleotide probes withinthe 5′ or 3′-termini of essential genes to show a hybridisation signalwith the TMDH protocol. For example, if a transposon insertion occurs ina non-essential gene adjacent to an essential gene, RNA target sequencesmay be generated from this transposon corresponding to bothnon-essential and essential gene sequences as a result of restrictionsites lying within the essential gene. The resulting labelled targetwill not only comprise DNA corresponding to the non-essential gene (thathas been disrupted), but will also extend into the adjacent essentialgene up to the restriction site. The result of hybridising this labelledtarget to the oligonucleotide array will be appear as “bleed through” ofsignal to probes on either the 5′ or 3′ end of the essential gene, up tothe restriction site used for the TMDH protocol.

To address this potential source of mis-assignment of essential genes,the restriction endonuclease digestion TMDH protocol described above maybe carried with more than one aliquot of the isolated genomic DNA, forexample two or three, whereby each aliquot is digested with a differentrestriction endonuclease (which have different recognition motifs). Themore aliquots digested, with more restriction sites that are used togenerate target sequences, the more statistically unlikely it is thatall of them will result in labelled RNA target sequences that “bleedthrough” into essential genes. The pools of RNA target sequences derivedfrom the different digestions can be hybridised to the same or,preferably, different oligonucleotide arrays if they were generatedusing different labels, or alternatively may be hybridised to copies ofthe same or, preferably, a different array. The analysis of theresulting multiple array hybridisation patterns will remove anyambiguity on the site of transposon insertion.

A different modification to the TMDH protocol, applicable to both thestandard restriction endonuclease and the homing endonucleaseapproaches, to minimize the mis-assignment of essential genes asnon-essential gene is to isolate two pools of RNA target sequences whicheach originate from different sides of the transposons. This approach isillustrated in FIGS. 5 and 6. FIGS. 5 and 6 show Gene Kelly transposonswhich comprise two RNA polymerase recognition sequences (although inFIG. 6 the two ends are shown superimposed).

In FIG. 5 one pool of RNA target sequences (from one side of thetransposons) is generated by carrying out in vitro transcription usingT7 polymerase and a second pool of RNA target sequences (from the otherside of the transposon) is generated by carrying out in vitrotranscription using T3 or SP6 polymerase.

Similarly, in FIG. 6 one pool of RNA target sequences (from one side ofthe transposons) is generated by carrying out in vitro transcriptionusing T7 polymerase and a second pool of RNA target sequences (from theother side of the transposon) is generated by carrying out in vitrotranscription using T3 or SP6 polymerase. The ends lying 5′ to the RNApolymerase recognition site (which are rescued using streptavidin coatedparticles) are generated via digestion with homing endonucleases. Thehoming endonuclease recognition site associated with each RNA polymeraserecognition site may be the same or different homing endonucleaserecognition sites.

The two pools of RNA target sequences can be hybridised to the sameoligonucleotide array if they were generated using different labels, oralternatively each may be hybridised to a separate copy of the samearray. Probes in the oligonucleotide array which shows no hybridisationto either pool are likely to correspond to essential genes.

Where an essential gene sequence is isolated in one of the RNA/cDNApools because it lies close to a non-essential gene sequence flanking atransposon insertion site, hybridisation to a probe on theoligonucleotide array will be observed even though that probecorresponds to an essential gene. However, that probe will show nohybridisation with the other pool of RNA target sequences which comprisethe flanking sequence from the other side of the transposon. Thus, inthis type of TMDH, a probe in the oligonucleotide array to which atleast one of the pools of RNA target sequences does not hybridise islikely to correspond to a gene that has not been disrupted by atransposon and may therefore be assigned as an essential gene.

In the methods described above (and also in those described below forthe identification of conditional essential genes), the pools of RNAsequences may be directly hybridised to arrays as set out above.Alternatively, before hybridisation is carried out, the pools of RNAsequences may be subjected to reverse transcription to generate pools ofcDNAs. The sequence of the particular transposon of the invention usedto generate the library of mutants may be used to design oligonucleotideprimers suitable for priming reverse transcription or, alternatively,random primers may be used. A label may be included in the reversetranscription reaction so that labelled cDNA pools are generated. ThecDNA pools may be then be hybridised to arrays as is described above inrelation to RNA pools.

The TMDH methods described above may also be used for the identificationof conditional essential genes. Conditional essential genes are thosewhich are not absolutely essential for bacterial survival, but areessential for survival in particular environments e.g. forgrowth/proliferation, in a host (in the case of a pathogenic bacterium)or for survival at elevated temperatures. Such environments aredescribed here as conditional restraints.

In order to isolate conditional essential genes, a library of transposonmutants is generated under control conditions (eg. growth at 37° C. incomplete media). The library of mutants is then subjected to someconditional restraint. For example, the library of mutants can beinoculated in a suitable host, if it is a pathogen. Alternatively, thelibrary of mutants can be grown at an elevated temperature. After thelibrary of mutants has been subjected to the conditional restraint itcan be recovered.

The library of mutants may be recovered by, for example, recoveringtissue such as the liver and/or spleen from a host and plating out anextract derived from that tissue on growth medium. All survivingcolonies on the plate represent mutants where a transposon insertion hasoccurred in a non-essential sequence. The surviving colonies can bepooled to give an output library of mutants which can then be subjectedto one of the TMDH methods set out above.

Alternatively, non-essential DNA sequences may be recovered directlyfrom tissue (such as the liver and/or spleen) of an infected hostwithout an intervening step of plating out an extract of the isolatedtissue. In this regard, the presence of one, or preferably two, homingendonuclease recognition sites in the transposon is crucial. Thus, thetissue may be subjected to digestion with a homing endonuclease forwhich there is a corresponding recognition site in the relevanttransposon. Typically, the tissue would be homogenised before digestion.Transposon ends (i.e. transposon:flanking sequence fragments) may thenbe recovered from the tissue. This may be achieved by annealingbiotinylated linkers to the transposon ends and recovering the resultingbiotinylated transposon ends with, for example, strepatavidin-coatedbeads. The isolated transposon:flanking sequence fragments may then bedigested with a restriction endonuclease which cuts in the sequenceflanking the transposon insertion site. Alternatively, the fragments maybe digested with a restriction endonuclease prior to recovery from thetissue.

The library of mutants that have been exposed to the conditionalrestraint will lack mutants which carry transposons in those genesessential for growth under the conditional environment, for examplegrowth/proliferation in a host organism.

The control and conditional restraint libraries can then be subjected tothe TMDH protocols described above using the a transposon of theinvention. Clearly, if transposon:flanking sequence fragments have beenisolated directly, for example from a host tissue using capture of,those fragments enter a TMDH protocol at the stage of preparing RNA bytranscription from the RNA polymerase site(s).

It is not necessary to use the same TMDH protocol for each library. Thetwo resulting RNA target sequence libraries may then be hybridisedseparately to high density oligonucleotide arrays. Alternatively theycan be hybridised to the same array, if the control and conditionalrestraint libraries are differentially labelled for example.

Comparison of the results given with the control and the conditionalrestraint libraries will allow the identification of genes which permitsurvival in the conditional restraint. Genes identified as essential forsurvival in the conditional restraint library, but not identified asessential for survival under control conditions should represent genesthat are essential for survival under the conditional restraintconditions. In particular, probes which show hybridisation with RNAtarget sequences from the input library but which show no hybridisationor substantially no hybridisation (there may be a low level ofbackground non-specific hybridisation) with RNA target sequences fromthe output library are strong candidates for sequences corresponding toconditional essential genes. The same “bleed through” considerationsapply as set out above and the modified TNDH protocols for overcomingsuch “bleed through” may need to be used.

In the case of the analysis of conditional mutations in a pathogen, alibrary of Salmonella typhimurium transposon mutants, for example, canbe used to infect a mouse. Following infection, bacteria target tolivers and spleens and the course of infection can be convenientlyfollowed by performing viable bacterial counts on those organs. Thebacteria recovered from the livers and spleens can be grown on suitableplates. In the case of the conditional restraint at elevatedtemperature, a transposon-tagged library can be grown at 42° C.

Other conditional restraints include growth of antibiotic resistantbacteria in the presence of antibiotics. This may reveal genes which areessential for antibiotic resistance. Such genes would be targets fordrugs with the ability to lower bacterial resistance to particularantibiotics. Organisms could be grown in the presence of carcinogens, UVor other agents that cause oxidative stress and thus genes that conferresistance to growth under those conditions may be identified.

Potential essential gene sequences and conditional essential genesequences identified by a TMDH strategy using a transposon of theinvention may be verified using a method based on allelic exchange. Thistechnique is particularly suitable for analysis of bacterial genes. PCRprimers can be used to generate left- and right-arm sequencescorresponding to the target gene sequence and ligated with akanamycin-resistance encoding gene cassette. The resulting cassette canbe introduced into a suicide vector, for example a plasmid-based vector,which is unable to replicate in a host bacterium.

In the case of a candidate essential gene, the resulting construct canbe introduced into the bacterial strain from which the candidate geneoriginates. If the target gene is essential, it should be impossible toisolate allelic-exchange mutants that have a disrupted version of thetarget gene. In the case of a candidate conditional essential gene, theessential gene can be introduced into the bacterial strain from whichthe candidate gene originates. Allelic-exchange mutants can be isolatedand subjected to growth under the conditional restraint. If thecandidate gene is a conditional essential gene, it should not bepossible for the allelic-exchange mutants to survive under theconditional restraint.

Similar experiments may be performed for other organisms.

The use of bioinformatics may allow the rapid isolation of furtheressential and conditional essential genes. A gene identified by TMDHusing a transposon of the invention may be used to search databasescontaining sequence information from other species in order to identifyorthologous genes from those species. Genes so identified can be testedfor being essential or conditionally essential using the genetictechniques described above. For example, an E. coli gene is identifiedas essential using a method as described above. This may allow theidentification of a putative orthologue from Salmonella. That Salmonellagene may be tested by allelic exchange and the construction ofconditional mutants in Salmonella as described above. Furtherorthologues may be identified in more distantly related organisms, forexample from Plasmodium species.

Suitable bioinformatics programs are well known to those skilled in theart. For example, the Basic Local Alignment Search Tool (BLAST) program(Altschul et al., 1990, J. Mol. Biol. 215, 403-410. and Altschul et al.,1997, Nucl. Acids Res. 25, 3389-3402.) may be used. Suitable databasesfor searching are for example, EMBL, GENBANK, TIGR, EBI, SWISS-PROT andtrEMBL.

Organisms that may be used in the invention are those for which it ispossible to carry out transposon mutagenesis and thus, those that cangive rise to a library of transposon mutants. Clearly, if the genome isbigger, more mutants will have to be produced in order to give a betterchance of achieving saturation mutagenesis. Suitable organisms includeprokaryotic and eukaryotic organisms. Suitable prokaryotes includebacteria Preferred bacteria are those which are animal or human or plantpathogens.

The bacteria used may be Gram-negative or Gram-positive. The bacteriamay be for example, from the genera Escherichia, Salmonella, Vibrio,Haemophilus, Neisseria, Yersinia, Bordetella, Brucella, Shigella,Klebsiella, Enterobacter, Serracia, Proteus, Vibrio, Aeromonas,Pseudomonas, Acinetobacter, Moraxella, Flavobacterium, Actinobacillus,Staphylococcus, Streptococcus, Mycobacterium, Listeria, Clostridium,Pasteurella, Helicobacter, Campylobacter, Lawsonia, Mycoplasma,Bacillus, Agrobacterium, Rhizobium, Erwinia or Xanthomonas. Examples ofsome of the above mentioned genera are Escherichia coli—a cause ofdiarrhoea in humans; Salmonella typhimurium—the cause of salmonellosisin several animal species; Salmonella typhi—the cause of human typhoidfever; Salmonella enteritidis—a cause of food poisoning in humans;Salmonella choleraesuis—a cause of salmonellosis in pigs; Salmonelladublin—a cause of both a systemic and diarrhoeal disease in cattle,especially of new-born calves; Haemophilus influenzae—a cause ofmeningitis; Neisseria gonorrhoeae—a cause of gonorrhoea; Yersiniaenterocolitica—the cause of a spectrum of diseases in humans rangingfrom gastroenteritis to fatal septicemic disease; Bordetellapertussis—the cause of whooping cough; Brucella abortus—a cause ofabortion and infertility in cattle and a condition known as undulantfever in humans; Vibrio cholerae—a cause of cholera, Clostridiumtetani—a cause of tetanus; Bacillus anthracis—a cause of anthrax.Suitable eukaryotes include fungi, plants and animals. Preferredeukaryotes include animal or human parasites and plant pests.

Suitable fungi include the animal pathogens including Candida albicans—acause of thrush, Trichophyton spp.—a cause of ringworm in children,athlete's foot in adults. Other suitable fungi include the plantpathogens Phytophthora infestans, Plasmopara viticola, Peronospora spp.,Saprolegnia spp., Erysiphe spp., Ceratocystis ulmi, Moniliniafructigena, Venturia inequalis, Claviceps purpurea, Diplocarpon rosae,Puccinia graminis, Ustilago avenae.

Suitable animal parasites include Plasmodium spp., Trypanasoma spp.,Giarda spp., Trichomonas spp. and Schistosoma spp. Other animalparasites include the various platyhelminth, nematode and annelidparasites.

Suitable plant pests include insects, nematodes and molluscs such asslugs and snails.

Suitable plants include monocotyledons and dicotyledons.

Preferred organisms are those for which the entire genome is known andfor which it may be possible to construct a high density oligonucleotidearray covering the entire genome or all of the open reading frames.

Essential and conditional essential genes, particularly essential genesare targets for drug discovery. That is, essential and conditionalessential genes of bacteria and the polypeptides which they encode mayrepresent targets for antibacterial substances, for example. Similarlyessential and conditional essential genes of fungi and eukaryoticparasites, pests and plants and the proteins which they encode mayrepresent targets for fungicides, antiparasitics, pesticides andherbicides respectively. Fungicides may have both animal and plantapplications. Additionally, conditional essential genes may representtargets for the generation of attenuated vaccines, particularly in thecase of bacterial conditional essential genes.

Furthermore, if a particular gene is essential or conditionallyessential for a number of different bacteria, fungi, parasites, pests orplants, that gene and the polypeptide it encodes may represent a targetfor substances with a broad-spectrum of activity.

An essential or conditional essential gene identified by one of themethods described above using a transposon of the invention and thepolypeptide which it encodes may be used in a method for identifying aninhibitor of transcription and/or translation of the gene and/oractivity of the polypeptide encoded by the gene. Such a method maycomprise identifying an essential or conditional essential gene using amethod of the invention and then determining whether a test substanceinhibits the transcription and/or translation of a gene thus identifiedor inhibits the activity of a polypeptide encoded by such a gene.

Such a substance may be referred to as an inhibitor of an essential orconditional essential gene. Thus, an inhibitor of an essential orconditional essential gene is a substance which inhibits expressionand/or translation of that essential gene and/or activity of thepolypeptide encoded by that essential or conditional essential gene.

Any suitable assay may be carried out to determine whether a testsubstance is an inhibitor of an essential or conditional essential gene.For example, the promoter of an essential or conditional essential genemay be linked to a coding sequence for a reporter polypeptide. Such aconstruct may be contacted with a test substance under conditions inwhich, in the absence of the test substance expression of the reporterpolypeptide would occur. This would allow the effect of the testsubstance on expression of the essential or conditional essential geneto be determined.

Substances which inhibit translation of an essential or conditionalessential gene may be isolated, for example, by contacting the mRNA ofthe essential or conditional essential gene with a test substance underconditions that would permit translation of the mRNA in the absence ofthe test substance. This would allow the effect of the test substance ontranslation of the essential or conditional essential gene to bedetermined.

Substances which inhibit activity of a polypeptide encoded by theessential gene may be isolated, for example, by contacting thepolypeptide with a substrate for the polypeptide and a test substanceunder conditions that would permit activity of the polypeptide in theabsence of the test substance. This would allow the effect of the testsubstance on activity of the polypeptide encoded by the essential orconditional essential gene to be determined.

Suitable control experiments can be carried out. For example, a putativeinhibitor should be tested for its activity against other promoters,mRNAs or polypeptides to discount the possibility that it is a generalinhibitor of gene transcription, translation or polypeptide activity.

Suitable test products which can be tested in the above assays includecombinatorial libraries, defined chemical entities, peptide and peptidemimetics, oligonucleotides and natural product libraries, such asdisplay (e.g. phage display libraries) and antibody products. Antibodyproducts include monoclonal and polyclonal antibodies, single chainantibodies, chimaeric antibodies and CDR-grafted antibodies.

Typically, organic molecules will be screened, preferably small organicmolecules which have a molecular weight of from 50 to 2500 daltons.Candidate products can be biomolecules including, saccharides, fattyacids, steroids, purines, pyrimidines, derivatives, structural analogsor combinations thereof. Candidate agents are obtained from a widevariety of sources including libraries of synthetic or naturalcompounds. Known pharmacological agents may be subjected to directed orrandom chemical modifications, such as acylation, alkylation,esterification, amidification, etc. to produce structural analogs.

Test substances may be used in an initial screen of, for example, forexample from 10 to 100 substances per reaction, and the substances ofthese batches which show inhibition or stimulation tested individually.Test substances may be used at a concentration of from 1 nM to 1000 μM,preferably from 1 μM to 100 μM, more preferably from 1 μM to 10 μM.Suitable test substances for inhibitors of essential or conditionalessential genes include combinatorial libraries, defined chemicalentities, peptides and peptide mimetics, oligonucleotides and naturalproduct libraries.

The test substances may be used in an initial screen of, for example,ten substances per reaction, and the substances of batches which showinhibition tested individually.

An inhibitor of an essential or conditional essential gene is one whichinhibits expression and/or translation of that essential gene and/oractivity of the polypeptide encoded by that essential or conditionalgene. Preferred inhibitors of the invention are those which inhibitessential gene expression and/or translation and/or activity by at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95% or at least99% at a concentration of the inhibitor of 1 ngml⁻¹, 10 ngml⁻¹, 100ngml⁻¹, 500 ngml⁻¹, 1 μgml⁻¹, 10 μgm⁻¹, 100 μgml⁻¹, 500 μgml⁻¹, 1mgml⁻¹, 10 mgml⁻¹, 100 mgml⁻¹. The percentage inhibition represents thepercentage decrease in expression and/or translation and/or activity ina comparison of assays in the presence and absence of the testsubstance. Any combination of the above mentioned degrees of percentageinhibition and concentration of inhibitor may be used to define aninhibitor of the invention, with greater inhibition at lowerconcentrations being preferred.

Test substances which show activity in assays such as those describedabove can be tested in in vivo systems, such as an animal model ofinfection for antibacterial activity or a plant model for herbicidalactivity. Thus, candidate inhibitors could be tested for their abilityto attenuate bacterial infections in mice in the case of anantibacterial or for their ability to inhibit growth of plants in thecase of a herbicide.

Inhibitors of bacterial, fungal or eukaryotic parasite essential orconditional essential genes may be used in a method of treatment of thehuman or animal body by therapy. In particular such substances may beused in a method of treatment of a bacterial, fungal or eukaryoticparasite infection. Such substances may also be used for the manufactureof a medicament for use in the treatment of a bacterial, fungal oreukaryotic parasite infections The condition of a patient suffering fromsuch an infection can be improved by administration of an inhibitor. Atherapeutically effective amount of an inhibitor may be given to a humanpatient in need thereof. Inhibitors of bacterial, fungal or eukaryoticparasite essential or conditional essential genes may be administered ina variety of dosage forms. Thus, they can be administered orally, forexample as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules. The inhibitors may also be administeredparenterally, either subcutaneously, intravenously, intramuscularly,intrasternally, transdermally or by infusion techniques. The inhibitorsmay also be administered as suppositories. A physician will be able todetermine the required route of administration for each particularpatient.

The formulation of an inhibitor for use in preventing or treating abacterial or fungal infection will depend upon factors such as thenature of the exact inhibitor, whether a pharmaceutical or veterinaryuse is intended, etc. An inhibitor may be formulated for simultaneous,separate or sequential use.

An inhibitor is typically formulated for administration in the presentinvention with a pharmaceutically acceptable carrier or diluent. Thepharmaceutical carrier or diluent may be, for example, an isotonicsolution. For example, solid oral forms may contain, together with theactive compound, diluents, e.g. lactose, dextrose, saccharose,cellulose, corn starch or potato starch; lubricants, e.g. silica, talc,stearic acid, magnesium or calcium stearate, and/or polyethyleneglycols; binding agents; e.g. starches, gum arabic, gelatin,methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone;disaggregating agents, e.g. starch, alginic acid, alginates or sodiumstarch glycolate; effervescing mixtures; dyestuffs; sweeteners; wettingagents, such as lecithin, polysorbates, laurylsulphates; and, ingeneral, non-toxic and pharmacologically inactive substances used inpharmaceutical formulations. Such pharmaceutical preparations may bemanufactured in known manner, for example, by means of mixing,granulating, tabletting, sugar-coating, or film-coating processes.

Liquid dispersions for oral administration may be syrups, emulsions orsuspensions. The syrups may contain as carriers, for example, saccharoseor saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain as carrier, for example a naturalgum, agar, sodium alginate, pectin, methylcellulose,carboxymethylcellulose, or polyvinyl alcohol. The suspensions orsolutions for intramuscular injections may contain, together with theactive compound, a pharmaceutically acceptable carrier, e.g. sterilewater, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and ifdesired, a suitable amount of lidocaine hydrochloride.

Solutions for intravenous administration or infusion may contain ascarrier, for example, sterile water or preferably they may be in theform of sterile, aqueous, isotonic saline solutions.

A therapeutically effective amount of an inhibitor is administered to apatient. The dose of an inhibitor may be determined according to variousparameters, especially according to the substance used; the age, weightand condition of the patient to be treated; the route of administration;and the required regimen. Again, a physician will be able to determinethe required route of administration and dosage for any particularpatient. A typical daily dose is from about 0.1 to 50 mg per kg of bodyweight, according to the activity of the specific inhibitor, the age,weight and conditions of the subject to be treated, the type andseverity of the degeneration and the frequency and route ofadministration. Preferably, daily dosage levels are from 5 mg to 2 g.

Conditional essential genes are good candidates for use in thepreparation of live attenuated vaccines. The principle behindvaccination is to induce an immune response in the host thus providingprotection against subsequent challenge with a pathogen. This may beachieved by inoculation with a live attenuated strain of the pathogen,i.e. a strain having reduced virulence such that it does not cause thedisease caused by the virulent pathogen. Bacteria which carry mutationsin conditional essential genes required for survival (i.e.growth/proliferation) in a host isolated according to the methodsdescribed above may be good candidates for use in live attenuatedvaccines.

Mutations introduced into a bacterium for use in a vaccine generallyknock-out the function of the conditional essential gene, for example agene required for growth/proliferation in a host, completely. This maybe achieved either by abolishing synthesis of any polypeptide at allfrom the gene or by making a mutation that results in synthesis ofnon-functional polypeptide. In order to abolish synthesis ofpolypeptide, either the entire gene or its 5′-end may be deleted. Adeletion or insertion within the coding sequence of a gene may be usedto create a gene that synthesises only non-functional polypeptide (e.g.polypeptide that contains only the N-terminal sequence of the wild-typeprotein).

The bacterium may have mutations in one or more, for example two, threeor four conditional essential genes. The mutations are non-revertingmutations. These are mutations that show essentially no reversion backto the wild-type when the bacterium is used as a vaccine. Such mutationsinclude insertions and deletions. Insertions and deletions arepreferably large, typically at least 10 nucleotides in length, forexample from 10 to 600 nucleotides. Preferably, the whole codingsequence is deleted.

The bacterium used in the vaccine preferably contains only definedmutations, i.e. mutations which are characterised. It is clearlyundesirable to use a bacterium which has uncharacterised mutations inits genome as a vaccine because there would be a risk that theuncharacterised mutations may confer properties on the bacterium thatcause undesirable side-effects.

The attenuating mutations may be introduced by methods well known tothose skilled in the art. Appropriate methods include cloning the DNAsequence of the wild-type gene into a vector, e.g. a plasmid, andinserting a selectable marker into the cloned DNA sequence or deleting apart of the DNA sequence, resulting in its inactivation. A deletion maybe introduced by, for example, cutting the DNA sequence usingrestriction enzymes that cut at two points in or just outside the codingsequence and ligating together the two ends in the remaining sequencewith an antibiotic resistance determinant. A plasmid carrying theinactivated DNA sequence can be transformed into the bacterium by knowntechniques such as electroporation or conjugation for example. It isthen possible by suitable selection to identify a mutant wherein theinactivated DNA sequence has recombined into the chromosome of thebacterium and the wild-type DNA sequence has been renderednon-functional by homologous recombination.

The attenuated bacterium of the invention may be genetically engineeredto express an antigen that is not expressed by the native bacterium (a“heterologous antigen”), so that the attenuated bacterium acts as acarrier of the heterologous antigen. The antigen may be from anotherorganism, so that the vaccine provides protection against the otherorganism. A multivalent vaccine may be produced which not only providesimmunity against the virulent parent of the attenuated bacterium butalso provides immunity against the other organism. Furthermore, theattenuated bacterium may be engineered to express more than oneheterologous antigen, in which case the heterologous antigens may befrom the same or different organisms. The heterologous antigen may be acomplete protein or a part of a protein containing an epitope. Theantigen may be from a virus, prokaryote or a eulcaryote, for exampleanother bacterium, a yeast, a fungus or a eukaryotic parasite. Theantigen may be from an extracellular or intracellular protein. Moreespecially, the antigenic sequence may be from E. coli, tetanus,hepatitis A, B or C virus, human rhinovirus such as type 2 or type 14,herpes simplex virus, poliovirus type 2 or 3, foot-and-mouth diseasevirus, influenza virus, coxsackie virus or Chlamydia trachomatis. Usefulantigens include non-toxic components of E. coli heat labile toxin,E.coli K88 antigens, ETEC colonization factor antigens, P.69 proteinfrom B. pertussis and tetanus toxin fragment C.

The DNA encoding the heterologous antigen is expressed from a promoterthat is active in vivo. Two promoters that have been shown to work wellin Salmonella are the nirB promoter and the htrA promoter. Forexpression of the ETEC colonization factor antigens, the wild-typepromoters could be used. A DNA construct comprising the promoteroperably linked to DNA encoding the heterologous antigen may be made andtransformed into the attenuated bacterium using conventional techniques.Transformants containing the DNA construct may be selected, for exampleby screening for a selectable marker on the construct. Bacteriacontaining the construct may be grown in vitro before being formulatedfor administration to the host for vaccination purposes.

The vaccine may be formulated using known techniques for formulatingattenuated bacterial vaccines. The vaccine is advantageously presentedfor oral administration, for example in a lyophilised encapsulated form.Such capsules may be provided with an enteric coating comprising, forexample, Eudragate “S” (Trade Mark), Eudragate “L” (Trade Mark),cellulose acetate, cellulose phthalate or hydroxypropylmethyl cellulose.These capsules may be used as such, or alternatively, the lyophilisedmaterial may be reconstituted prior to administration, e.g. as asuspension. Reconstitution is advantageously effected in a buffer at asuitable pH to ensure the viability of the bacteria In order to protectthe attenuated bacteria and the vaccine from gastric acidity, a sodiumbicarbonate preparation is advantageously administered before eachadministration of the vaccine. Alternatively, the vaccine may beprepared for parenteral administration, intranasal administration orintramuscular administration.

The vaccine may be used in the vaccination of a mammalian host,particularly a human host but also an animal host. An infection causedby a microorganism, especially a pathogen, may therefore be prevented byadministering an effective dose of a vaccine prepared according to theinvention. The dosage employed will ultimately be at the discretion ofthe physician, but will be dependent on various factors including thesize and weight of the host and the type of vaccine formulated. However,a dosage comprising the oral administration of from 10⁷ to 10¹¹ bacteriaper dose may be convenient for a 70 kg adult human host.

Inhibitors of bacterial, fungal and pest essential or conditionalessential genes may be administered to plants in order to prevent ortreat bacterial, fungal or pest infections; the term pest includes anyanimal which attacks a plant. Thus inhibitors of the invention may beuseful as pesticides. Inhibitors of plant essential or conditionalessential genes may be administered to plants in order to reduce or stopplant growth, that is to act as a herbicide.

The inhibitors of the present invention are normally applied in the formof compositions together with one or more agriculturally acceptablecarriers or diluents and can be applied to the crop area or plant to betreated, simultaneously or in succession with further compounds.

The inhibitors of the invention can be selective herbicides,bacteriocides, fungicides or pesticides or mixtures of several of thesepreparations, if desired together with further carriers, surfactants orapplication-promoting adjuvants customarily employed in the art offormulation. Suitable carriers and diluents correspond to substancesordinarily employed in formulation technology, e.g. natural orregenerated mineral substances, solvents, dispersants, wetting agents,tackifiers, binders or fertilizers.

A preferred method of applying active ingredients of the presentinvention or an agrochemical composition which contains at least one ofthe active ingredients is leaf application. The number of applicationsand the rate of application depend on the intensity of infestation bythe pathogen. However, the active ingredients can also penetrate theplant through the roots via the soil (systemic action) by impregnatingthe locus of the plant with a liquid composition, or by applying thecompounds in solid form to the soil, e.g. in granular form (soilapplication). The active ingredients may also be applied to seeds(coating) by impregnating the seeds either with a liquid formulationcontaining active ingredients, or coating them with a solid formulation.In special cases, further types of application are also possible, forexample, selective treatment of the plant stems or buds.

The active ingredients are used in unmodified form or, preferably,together with the adjuvants conventionally employed in the art offormulation, and are therefore formulated in known manner toemulsifiable concentrates, coatable pastes, directly sprayable ordilutable solutions, dilute emulsions, wettable powders, solublepowders, dusts, granulates, and also encapsulations, for example, inpolymer substances. Like the nature of the compositions, the methods ofapplication, such as spraying, atomizing, dusting, scattering orpouring, are chosen in accordance with the intended objectives and theprevailing circumstances. Advantageous rates of application are normallyfrom 50 g to 5 kg of active ingredient (a.i.) per hectare (“ha”,approximately 2.471 acres), preferably from 100 g to 2 kg a.i./ha, mostpreferably from 200 g to 500 g a.i./ha.

The formulations, compositions or preparations containing the activeingredients and, where appropriate, a solid or liquid adjuvant, areprepared in known manner, for example by homogeneously mixing and/orgrinding active ingredients with extenders, for example solvents, solidcarriers and, where appropriate, surface-active compounds (surfactants).

Suitable solvents include aromatic hydrocarbons, preferably thefractions having 8 to 12 carbon atoms, for example, xylene mixtures orsubstituted naphthalene, phthalate such as dibutyl phthalate or dioctylphthalate, aliphatic hydrocarbons such as cyclohexane or paraffins,alcohols and glycols and their ethers and esters, such as ethanol,ethylene glycol, monomethyl or monoethyl ether, ketones such ascyclohexanone, strongly polar solvents such as N-methyl-2-pyrrolidone,dimethyl sulfoxide or dimethyl formamide, as well as epoxidizedvegetable oils such as epoxidized coconut oil or soybean oil; or water.

The solid carriers used e.g. for dusts and dispersible powders, arenormally natural mineral fillers such as calcite, talcum, kaolin,montmorillonite or attapulgite. In order to improve the physicalproperties it is also possible to add highly dispersed silicic acid orhighly dispersed absorbent polymers. Suitable granulated adsorptivecarriers are porous types, for example pumice, broken brick, sepioliteor bentonite; and suitable nonsorbent carriers are materials such ascalcite or sand. In addition, a great number of pregranulated materialsof inorganic or organic nature can be used, e.g. especially dolomite orpulverized plant residues.

Depending on the nature of the active ingredient to be used in theformulation, suitable surface-active compounds are nonionic, cationicand/or anionic surfactants having good emulsifying, dispersing andwetting properties. The term “surfactants” will also be understood ascomprising mixtures of surfactants. Suitable anionic surfactants can beboth water-soluble soaps and water-soluble synthetic surface-activecompounds.

Suitable soaps are the alkali metal salts, alkaline earth metal salts orunsubstituted or substituted ammonium salts of higher fatty acids(chains of 10 to 22 carbon atoms), for example the sodium or potassiumsalts of oleic or stearic acid, or of natural fatty acid mixtures whichcan be obtained for example from coconut oil or tallow oil. The fattyacid methyltaurin salts may also be used.

More frequently, however, so-called synthetic surfactants are used,especially fatty sulfonates, fatty sulfates, sulfonated benzimidazolederivatives or alkylarylsulfonates.

The fatty sulfonates or sulfates are usually in the form of alkali metalsalts, alkaline earth metal salts or unsubstituted or substitutedammoniums salts and have a 8 to 22 carbon alkyl radical which alsoincludes the alkyl moiety of alkyl radicals, for example, the sodium orcalcium salt of lignonsulfonic acid, of dodecylsulfate or of a mixtureof fatty alcohol sulfates obtained from natural fatty acids. Thesecompounds also comprise the salts of sulfuric acid esters and sulfonicacids of fatty alcohol/ethylene oxide adducts. The sulfonatedbenzimidazole derivatives preferably contain 2 sulfonic acid groups andone fatty acid radical containing 8 to 22 carbon atoms. Examples ofalkylarylsulfonates are the sodium, calcium or triethanolamine salts ofdodecylbenzenesulfonic acid, dibutylnaphthalenesulfonic acid, or of anaphthalenesulfonic acid/formaldehyde condensation product. Alsosuitable are corresponding phosphates, e.g. salts of the phosphoric acidester of an adduct of p-nonylphenol with 4 to 14 moles of ethyleneoxide.

Non-ionic surfactants are preferably polyglycol ether derivatives ofaliphatic or cycloaliphatic alcohols, or saturated or unsaturated fattyacids and alkylphenols, said derivatives containing 3 to 30 glycol ethergroups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moietyand 6 to 18 carbon atoms in the alkyl moiety of the alkylphenols.

Further suitable non-ionic surfactants are the water-soluble adducts ofpolyethylene oxide with polypropylene glycol, ethylenediamine propyleneglycol and alkylpolypropylene glycol containing 1 to 10 carbon atoms inthe alkyl chain, which adducts contain 20 to 250 ethylene glycol ethergroups and 10 to 100 propylene glycol ether groups. These compoundsusually contain 1 to 5 ethylene glycol units per propylene glycol unit.

Representative examples of non-ionic surfactants arenonylphenolpolyethoxyethanols, castor oil polyglycol ethers,polypropylene/polyethylene oxide adducts,tributylphenoxypolyethoxyethanol, polyethylene glycol andoctylphenoxyethoxyethanol. Fatty acid esters of polyoxyethylene sorbitanand polyoxyethylene sorbitan trioleate are also suitable non-ionicsurfactants.

Cationic surfactants are preferably quaternary ammonium salts whichhave, as N-substituent, at least one C₈-C₂₂ alkyl radical and, asfurther substituents, lower unsubstituted or halogenated alkyl, benzylor lower hydroxyalkyl radicals. The salts are preferably in the form ofhalides, methylsulfates or ethylsulfates, e.g. stearyltrimethylammoniumchloride or benzyldi(2-chloroethyl)ethylammonium bromide.

The surfactants customarily employed in the art of formulation aredescribed, for example, in “McCutcheon's Detergents and EmulsifiersAnnual”, MC Publishing Corp. Ringwood, N.J., 1979, and Sisely and Wood,“Encyclopaedia of Surface Active Agents,” Chemical Publishing Co., Inc.New York, 1980. The agrochemical compositions usually contain from about0.1 to about 99% preferably about 0.1 to about 95%, and most preferablyfrom about 3 to about 90% of the active ingredient, from about 1 toabout 99.9%, preferably from about 1 to 99%, and most preferably fromabout 5 to about 95% of a solid or liquid adjuvant, and from about 0 toabout 25%, preferably about 0.1 to about 25%, and most preferably fromabout 0.1 to about 20% of a surfactant.

Whereas commercial products are preferably formulated as concentrates,the end user will normally employ dilute formulations.

The following Examples illustrate the invention:

EXAMPLES

Materials and Methods

Unless indicated otherwise, the methods used are standard biochemicaltechniques. Examples of suitable general methodology textbooks includeSambrook et al., Molecular Cloning, a Laboratory Manual (1989) andAusubel et al., Current Protocols in Molecular Biology (1995), JohnWiley & Sons, Inc.

E. coli strains were grown on L-Agar (Sigma) and in L-Broth (Sigma).Kanamycin was purchased from Sigma S. typhimurium SL1344 was grown inTryptic Soy Broth (TSB, Oxoid).

Example 1 Construction of a Gene-Kelly Transposon

The EZ:Tn R6k ori Kan transposon (Epicentre) was used as a PCR templatewith oligonucleotides: (SEQ ID NO: 2) 97 5′-CAGCTGTCTCTTATACACATCTCCCTATAGTGAGTCGTATT AC CCATAATACCCATAATAGCTGTTTGCCAgtcgactctagagg-3′;and (SEQ ID NO: 3) 97 5′-CAGCTGTCTCTTATACACATCTCTTCTATAGTGTCACCTAAA TAGGGATAACAGGGTAATGaattcgttaatacagatgt-3′.

ME are italicised, the RNA polymerase binding sites are in bold, and thehoming endonuclease sites are underlined. These oligonucleotidesincorporate the T7 RNA polymerase site with the homing endonuclease sitePI-PspI, and the SP6 RNA polymerase site with the homing endonucleasesite for I-SceI, respectively. PCR was carried out according to protocolfor Roche Expand Hi-fidelity Kit.

A first round of PCR, to introduce the homing endonuclease and RNApolymerase sites, was carried out with an initial denaturation step at96° C. for 3 min, then 5 cycles of 96° C. 30s, 25° C. 90s, 72° C. 2 min30s, then a further 25 cycles at 96° C. 30s, 50° C. 90s, 72° C. 2 min30s, final extension of 4 minutes and then cooled to 4° C. The PCRproduct was separated by gel electrophoresis, and the band(corresponding to the expected product at 2044 bp) cut out and Gelextracted according to the Qiagen gel extraction protocol. The PCRproducts were re-ligated (according to Gibco protocol for T4 DNA ligase)overnight at 16° C. The ligation mixture was then purified using theQiagen Gel Extraction protocol and the circularised transposon eluted in50 μl water. 1 μl of the Transposon DNA was then electroporated into 40μl TransforMax™ EC100D™ pir⁺ Electrocompetent E. coli (Epicentre; using0.1 cm cuvettes at 200Ω, 25 μF and 20 KV/cm), outgrowth was in 1 ml SOCmedium (Gibco life technologies) for 1 h at 37° C. The transformationwas then plated out on L-Agar (Sigma) plates containing Kanamycin at 30μg/ml and incubated overnight at 37° C. 12 colonies were then picked andgrown up overnight in LB-Kan 30 μg/ml and 1.5 ml of culture was used forDNA-mini-preps according to the Qiagen protocol. The DNA sequence of theresulting constructs was then determined on a Beckman CEQ DNA sequencer,(using manufacturers recommended conditions) with kan-2-FP-1 forwardprimer (Epicentre) as a DNA sequencing primer. The sequence of clone11(GK11) was found to contain a single base pair change in one of the MEsequences. To correct this sequence change, an experiment using a secondround of PCR was designed with an oligonucleotide that corrected thesingle base pair change. The clone GK11 (harbouring a single-pointmutation in the ME) was re-PCR'd using un-phosphorylated ME primers (96°C. for 3 min, followed by 30 cycles of 96° C. for 30s, 45° C. for 90s,72° C. for 150s). The PCR products were then cloned into pBAD-TOPO(Invitrogen) and transformed into E. coli TOP10 chemically competentcells according to the Invitrogen protocol. The transformation mix wasplated out on L-Agar containing kanamycin at 30 μg/ml and incubatedovernight at 37° C. 6 colonies were then picked and grown in L-Brothcontaining kanamycin at 30 μg/ml overnight at 37° C. DNA from 1.5 mLsamples of these cultures was then prepared using the miniprep method(Qiagen QIAprep spin miniprep kit) according to the Qiagen protocol. TheDNA sequences of the inserts from each of these 6 minipreps was thendetermined with oligonucleotides kan-2-FP-1 and R6kan-2-RP-1 (Epicentre)as sequencing primers according to Beckman CEQ protocol. One of theisolates, clone 6A was correct when sequenced, named pBAD-GK6A, and wasselected for further study.

Example 2 Evaluation of the Gene-Kelly Transposon

(i) T7 and SP6 RNA Polymerase Evaluation

In order to evaluate the novel transposon generated in Example 1, 0.2 μgof pBAD-GK6A was used as template in an in vitro transcription (TVT)reactions using both SP6 (SP6 Megascript kit; Ambion) and T7 (T7Megashortscipt kit, Ambion) RNA polymerases, according to Ambionprotocols. RNA produced was purified using the Qiagen RNeasy mini Kit,and the amount of RNA produced measured at A₂₆₀. Transcription wasobserved both with the SP6 and T7 IVT reactions.

(ii) Restriction Digest of Transposon DNA with Homing Endonucleases

In order to determine whether or not the homing endonuclease sites werefunctional in the GK transposon, plasmid DNA (0.5 μg) was digested withI-SceI and PI-PspI. Separation of the resulting DNA products by agarosegel electrophoresis showed a single linearised band of the correct size.

(iii) Transposition of Salmonella typhimurium

pBAD-GK6A (0.1 μg) was electroporated into 40 μl S. typhimurium SL1344electrocompetent cells (SL1344 was grown to an OD 0.5 in 100 mltryptic-soy broth (TSB from Oxoid) at 37° C. Cells were centrifuged at5000×g for 10 min at 4° C., washed three times in 50 ml 10% glycerolbefore a final re-suspension in 1 ml of 10% glycerol) using a 0.2 cmcuvette, 2000, 25 μF and 12 kV/cm, outgrowth was in 1 ml SOC (Gibco) at37° C. for 1 h, the transformation was then plated onto L-Agar platescontaining Kanamycin at 50 μg/ml plates and grown overnight at 37° C. Acolony was then picked and grown in 2.5 litres L-Broth containingKanamycin at 50 μg/ml overnight at 37° C. A Qiagen Qiafilter Megaplasmid kit was used to purify pBAD-GK6A.

Example 3 Generation of S. typhimurium SL1344 Mutants with TransposonGK6a

The transposome complex was generated from pBAD-GK6A as follows.pBAD-GK6a (100 μg) of was digested with XmnI and NcoI (NEB) in NEBbuffer 2 (with 1 μg BSA/ml), overnight at 37° C. The entire digest wasthen run out on a 0.8% agarose gel, the ˜2 kb band, the GK transposon,was gel extracted using the Qiagen gel extraction kit, and eluted from 1spin column in 50 μl TE pH8.5. The GK transposome complex was generatedaccording to Epicentre protocols and electroporated intoelectrocompetent S. typhimurium SL1344 cells as described above.Following outgrowth the cells were subsequently plated out on TrypticSoy Agar plates (TSA, Oxoid) containing Kanamycin at 50 μg/ml andincubated overnight at 37° C. A total of 480 mutants were picked andgrown overnight in 2 ml TSB (Oxoid) containing Kanamycin at 50 μg/ml andglycerol stocks made (20% glycerol TSB).

Example 4 Recovery of Transposon for Sequencing (use of R6k Origin ofReplication)

Transposons and adjacent flanking DNA, corresponding to genes that havebeen disrupted by transposon insertion can be recovered from mutantchromosomal DNA samples using the use of R6k origin of replication.Digestion of chromosomal DNA purified from S. typhimurium SL1344 GKmutants with a restriction enzyme that does not cut in the transposon,followed by circularising the fragments, and transformation into a pir⁺strain of E. coli, results in the “rescuing” of this DNA.

Mutants 1-50 were grown up individually in 2 ml TSB containing Kanamycinat 50 μg/ml overnight at 37° C. Samples (1.5 ml) was used to preparechromosomal DNA using the Qiagen DNeasy tissue kit. A total of 5 μl (0.5μg) of each of the fifty chromosomal preps was digested with EcoRV (NEB)in a final volume of 20 μl, overnight at 37° C. The EcoRV was heatinactivated at 80° C. for 20 minutes. The 20 μl digest was thenreligated in 100 μl final volume using Gibco T4 DNA ligase, 48 hours at4° C. Each religation was then individually cleaned up using a Qiagengel extraction spin column and eluted in 50 μl water. Ligations (4 μl,0.04 μg) were electroporated into electrocompetent pir⁺ E. coli (EC100D,Epicentre) according to Epicentre protocols and plated on L-Agarcontaining Kanamycin at 30 μg/ml and incubated overnight at 37° C.Colonies were obtained from 46 of the electroporations, and weresubsequently grown up in 5 ml L-Broth containing Kanamycin at 30 μg/ml37° C. overnight Plasmid DNA (2 μg) from these clones was sequencedaccording to the Beckman CEQ protocol using oligonucleotide 108 as asequencing primer (T7 end of the transposon).

Example 5 Generation of RNA Run-Offs Using iPCR and IVT for TargetHybridisation to Microarrays

The generation of labelled target from GK mutants can be achieved byinverse PCR (iPCR) amplification of each end of the transposon followedby IVT reactions using either SP6 or T7 RNA polymerises. A pool of 96 S.typhimurium SL1344 mutants was inoculated into L-Broth (10 ml)containing Kanamycin at 50 μg/ml and grown overnight at 37° C.statically. Chromosomal DNA (20 μg) was prepared from 1.5 ml of cultureusing the Qiagen DNeasy Kit, and 5 μl (0.5 μg) digested individuallywith the restriction enzymes HaeIII, HhaI, Hpych4 IV and RsaI (NEB) intheir respective NEB buffers in a final volume of 20 μl, overnight at37° C. The enzymes were then heat denatured at 65° C. for HhaI, Hpych4IV and RsaI and 80° C. for HaeIII for 20 min. Each 20 μl digest was thenself-ligated with T4 DNA ligase (Gibco) in a 100 μl reaction at 4° C.for 48 h. Amplification of the DNA flanking each end of the transposonwas achieved by iPCR. iPCR reactions to amplify the SP6 end of thetransposon are performed with: oligonucleotide 107 (SEQ ID NO: 4)5′-CTACCCTGTGGAACACCTACATCT-3′; and one of either oligonucleotide 115(SEQ ID NO: 5) 5′-ATTACCTCTTTCTCCGCACCCGAC-3′; RsaI or Hpych4IV oroligonucleotide 116 (SEQ ID NO: 6) 5′-CGACATAGATCCGGAACATAATGG-3′;HaeIII or HhaI, depending on the restriction enzyme used (in brackets)to cut the chromosomal DNA. iPCR reactions to amplify the T7 end of thetransposon were performed with oligonucleotide 108 (SEQ ID NO: 7)5′-ACCTACAACAAAGCTCTCATCAACC-3′ and one of either oligonucleotide 117(SEQ ID NO: 8) 5′-ACAACCTATTAATTTCCCCTCGTC-3′; RsaI, HaeIII or BhaI oroligonucleotide 118 (SEQ ID NO: 9) 5′-ATGTTGGAATTTAATCGCGGCCTC-3′;Hpych4IV, depending on the restriction enzyme used (in brackets) to cutthe chromosomal DNA. iPCR reactions were then set up using Qiagen Taqpolymerase according to the Qiagen protocol. 4 μl (0.02 μg) of ligationwas used as template for each iPCR reaction. The reactions wereinitially denatured at 94° C. 3′ followed by 30 cycles of 94° C. 30s,65° C. 90s, 72° C. 90s followed by 7 min extension at 72° C. and thencooling to 4° C. Each of the 8 iPCR's were purified using a Qiagen Gelextraction kit and the DNA eluted in 5 μl water. Each iPCR product wasthen re-digested with its respective restriction enzyme in a finalvolume of 50 μl (NEB) overnight 37° C. The digests were then cleanedusing a Qiagen Gel extraction kit (following the manufacturersrecommended procedure) and the DNA eluted in 50 μl EB pH8.5.

Each digested iPCR product (2 μl) was used as a template for both T7 andSP6 in vitro transcription reactions according to the Ambion protocol.The RNA was cleaned using the Qiagen Rneasy kit and eluted in 50 μl ofRNase free water that was then placed in a UV transparent 96 well plateand the absorbance at 260 nm measured.

Example 6 Ligation Capture Recovery of Gene Kelly Transposon Ends

A significant advantage of the Gene Kelly (GK) transposon is that itpermits the recovery of DNA fragments adjacent to the site of transposoninsertion by a method that does not employ PCR, which is ligationcapture. Essentially, because of the rarity of the I-SceI and PI-PspIhoming endonuclease sites the T7 and SP6 promoter sites that are linkedto these sites can be enriched from a pool of DNA by ligation of abiotinylated linker to the cut site followed by purification usingStreptavidin linked magnetic beads. A ligation capture experiment wasperformed on pBAD-GK6A. Plasmid DNA (1 μg) was digested with PI-PspIovernight and the resulting linearised DNA purified using a Qiagen Gelextraction kit. This DNA (400 ng) was then digested overnight withHaeIII and subsequently dephosphorylated using Calf Intestinal AlkalinePhosphatase (Roche). Dephosphorylated DNA was then ligated overnightonto a biotinylated linker, generated by annealing oligonucleotide 113(SEQ ID NO: 10) 5 ′-biotin-GACGACCTCAGTTACGGTACGATCGGCCACGTAGCTTAT-3′and oligonucleotide 114 (SEQ ID NO: 11)5′-phosphate-GCTACGTGGCCGATCGTACCGTAACTGAGGTCGTC-3′.

The ligation was purified using a Qiagen Gel extraction. BiotinylatedDNA was then extracted from the ligation using Streptavidin-linkedmagnetic particles (150 μg; Promega) according to the manufacturersprotocol, and the beads finally resuspended in 8 μl of 1×HaeIIIrestriction buffer containing 10 units of HaeIII (NEB). The digestionwas then incubated at 37° C. for 2 hours to remove the T7 RNA promoterfrom the linker. The beads were removed and an IVT reaction performed onthe supernatant using an Ambion T7 Megashortscript kit, according to themanufacturers instructions. IVT products were purified using a QiagenRNeasy kit, and the products eluted in 50 μl water and read at A₂₆₀.

From the sequence data obtained we were able to identity the transposoninsertion point in the published Salmonella genome LT2. FIG. 4 shows agraph showing the random distribution of the sites of GK transposondistribution in the LT2 genome for the 46 sequenced mutants.

Example 7 Identification of Genes in Salmonela typhimurium Important forVirulence in the Mouse Model of Infection

480 random Gene Kelly transposon mutants were generated in Salmonellatyphimurium SL1344. The mutants were grown individually in 1 ml ofL-broth in 96-well HTS plates, overnight and subsequently pooled.Chromosomal DNA (78.6 μg) was purified and subsequently restrictiondigested separately with HaeIII and RsaI and the products purified withQiagen gel extraction columns. The DNA was divided in half and separateIVT reactions (19.6 μg DNA templates/IVT) were performed with therespective RNA polymerase (SP6 and T7) resulting in the generation oflabelled target (SP6 and T7 target was labelled with Cy5 and Cy3,respectively) corresponding to the DNA flanking both ends of transposoninsertions.

DNA microarrays were designed based on the entire Salmonella typhimuriumLT2 genome sequence, with probes synthesised in both the sense andanti-sense directions. Following hybridisation, the data were extractedfrom the arrays and analysed to identify those genes disrupted bytransposon insertion within the pool of 480 mutants. New microarrayswere designed incorporating probes corresponding to each site oftransposon insertion.

The mutants were grown individually in 1 ml of L-broth in 96-well HTSplates, overnight. Cultures were pooled (‘Input pool’) and chromosomalDNA purified from 50 ml of the resultant culture (‘Input pool’ DNA). Aninoculum of 10⁵ cfu/ml PBS was generated from the ‘input pool’. Micewere inoculated i.v. with 10⁵ cfu of the ‘input pool’ and the infectionallowed to proceed for 2.5 days, whereupon the mice were sacrificed andliver and spleen removed. Organs were pulverised in 10 ml water andspread onto 4×120 mm diameter L-Agar plates and incubated overnight at37° C. Bacteria (‘output pool’) were harvested from these plates byre-suspending the bacterial lawns in 10 ml L-broth/plate. ChromosomalDNA (‘output pool DNA’) was purified from 3 ml of this suspension.

‘Input’ and ‘output pool’ DNA (5 μg) was digested overnight with therestriction endonuclease RsaI, and subsequently cleaned on a Qiagen Gelextraction column. In vitro transcription reactions were set up usingthe Ambion T7 and SP6 Megascript kits, using 2 μg of digested DNA astemplate, with Cy3-CTP and Cy5-CTP, respectively, and incubated at 37°C. for 24 hrs. Following the DNaseI step RNA was purified using a Qiagennucleotide removal kit The RNA from both the SP6 and T7 IVT reactionswere then hybridised overnight to a DNA microarray containing probescorresponding to each transposon insertion site. Arrays were washed in0.6×SSPE X followed by 0.06×SSPE 0.18% PEG200, each wash lasting 5 min.Slides were then dried and analysed using an Agilent microarray scanner.Several mutants in the pool of 96 were analysed using DNA sequencing toascertain the precise point of transposition within the SL1344chromosome. One mutant was characterised as containing a transposonwithin the aroA gene that would lead to the loss of its function. AroAmutants are one of the best genetically defined S. typhimurium vaccinestrains (Chatfield et al., 1992; Microb. Pathog 12: 145-151). Thesemutants are reduced in their ability to survive within susceptible micecompared to its wild type parent strain, such that 3 days post infectionbacterial levels are reduced 1000-fold.

Data from the microarray images were extracted using Agilent's imageanalysis software and the data fed into a data viewing package. Arraydata from the input and output hybridisations were compared (see FIG.7).

Analysis of both the array image and the extracted data from the inputpool reveal that target was generated to the aroA gene and that thistarget hybridised to the expected probes surrounding the site oftransposon insertion. Analysis of the corresponding data from all threeoutput pools revealed that significantly less target was hybridised tothe probes corresponding to the aroA gene compared to two controlmutants with transposons disrupting other loci (gene X and an intergenicregion) in the SL1344 genome. These data indicate that the aroA mutantis attenuated in its ability to survive within the mice. Therefore thistechnique allows the identification of genes that are important for thevirulence of S. typhimurium in the mouse model of infection.

Example 8 Construction of a Mariner Erm Gene Kelly Transposon

The Gene Kelly R6k ori Kan transposon (i.e. the transposon generated inExample 1) was used as a PCR template with oligonucleotides: 135 (SEQ IDNO: 12) 5′-TAACAGGTTGGCTGATAAGTCCCCGGT CTCCC TATAGTGAG-3′; and 136 (SEQID NO: 13) 5′-TAACAGGTTGGCTGATAAGTCCCCGGT C T C TTCTATAGTGTC-3′.

Insertion sequences are italicised, overlap with the RNA polymerasebinding sites present in the Tn5 Gene Kelly construct are in bold. PCRwith these oligonucleotides maintain the internal design of Tn5 GeneKelly transposon comprising the T7 RNA polymerase site adjacent to thehoming endonuclease site PI-PspI, and the SP6 RNA polymerase siteadjacent to the homing endonuclease site for I-SceI, respectively. PCRwas carried out according to protocol for Roche Expand Hi-fidelity Kit.

A first round of PCR, to ensure annealing of the short oligonucleotideoverlap sequences with the denatured Tn5 Gene Kelly, was carried outwith an initial denaturation step at 94° C. for 3 min, then 5 cycles of94° C. 30s, 40° C. 90s, 72° C. 2 min 30s, then a further 25 cycles at94° C. 30s, 55° C. 90s, 72° C. 2 min 30s, final extension of 5 minutesand then cooled to 4° C. The PCR product was separated by gelelectrophoresis, and the band (corresponding to the expected product at2064 bp) cut out and Gel extracted according to the Qiagen gelextraction protocol. The PCR products were ligated (according to Gibcoprotocol for T4 DNA ligase) into the EcoRV restriction site of pETBlue-1(Novagen), overnight at 16° C. The ligation mixture was then transformedinto E.coli TOP10 cells (Invitrogen). The transformation was then platedout on L-Agar (Sigma) plates containing Kanamycin at 30 μg/ml andincubated overnight at 37° C. 12 colonies were then picked and grown upovernight in LB-Kan 30 μg/ml and 1.5 ml of culture was used forDNA-mini-preps according to the Qiagen protocol. The DNA sequence of theresulting constructs was then determined on a Beckman CEQ DNA sequencer,(using manufacturers recommended conditions) with the supplied pETBlue-1Down and Up sequencing primers. The sequence of clone 5 pETBlueHGK5) wasfound to contain the expected DNA sequence.

Example 9 Cloning of an Erythromycin Resistance Marker into Mariner ErmGene Kelly

A plasmid containing an erythromycin resistance marker, originally fromplasmid pIL253 (Simon & Chopin (1988), Biochimie 70 (4), 559-566), wascloned into pETBlueHGK5 to provide a selection marker for the Transposonfollowing integration into the chromosome of selected Gram-positivebacteria The erythromycin gene was amplified from pIL253 using primers:

-   -   5′ erm 5′-GATATCGAAGCAAACTTAAGAGTGT-3′ (SEQ ID NO: 14)    -   3′ erm 5′-GATATCTACAAAAGCGACTCATAGA-3′ (SEQ ID NO: 15)        and cloned into pCR2.1 Invitrogen). The EcoRV restriction sites        (underlined) were used to remove the erythromycin cassette from        this vector following digestion with the respective restriction        enzyme (NEB). The resistance marker (871 bp) was then purified        by agarose gel electrophoresis followed by the Qiagen gel        extraction protocol. The plasmid pETBlueHGK5 was linearised        (5540 bp) with the restriction enzyme HincII (NEB), and        dephosphorylated using Shrimp Alkaline phosphatase (Roche) and        then purified by agarose gel electrophoresis followed by the        Qiagen gel extraction protocol. The products were ligated        (according to Gibco protocol for T4 DNA ligase) overnight at        16° C. The ligation mixture was then transformed into E.coli        TOP10 cells (Invitrogen). The transformation was then plated out        on L-Agar (Sigma) plates containing Kanamycin at 30 μg/ml and        erythromycin at 200 μg/ml and incubated overnight at 37° C. 6        colonies were then picked and grown up overnight in LB-Kan 30        μg/ml and 1.5 ml of culture was used for DNA-mini-preps        according to the Qiagen protocol. The DNA sequence of the        resulting constructs was then determined on a Beckman CEQ DNA        sequencer, (using manufacturers recommended conditions) with the        sequencing primers:    -   12 5′-AAG ATA CTG CAC TAT CAA CAC ACT C-3′ (SEQ ID NO: 16)    -   13 5′-ATT AAG AAG GAG TGA TTA CAT GAA C-3′ (SEQ ID NO: 17)        as well as the pETBlue-1 Down and Up sequencing primers        (Novagen).

The sequence of clone 3 (pHGK5erm3) was found to contain the desired DNAsequence. The transposon contained within this plasmid was named theMariner Erm Gene Kelly transposon.

Example 10 Mutagenesis of Staphylococcus aureus with the Mariner ErmGene Kelly

A protocol (see below; Generation of a Mariner Erm Gene Kelly transposonlibrary) and strains for mutagenesis of Staphylococcus aureus wasobtained from Prof. S. Foster (Sheffield University, MTA). The protocolresults in Mariner Transposon integration into the S. aureus chromosome,following introduction of two temperature sensitive plasmids into therecipient strain (one bearing the Mariner Transposon; TS1, the other theMariner Transposase gene; pSPT246), and induction of transposition.

Example 11 Construction of S. aureus Strains Replicating Mariner ErmGene Kelly

Plasmid TS1 was digested with the restriction endonuclease BamHI (NEB),dephosphorylated with alkaline phosphatase (Roche) and purified byagarose gel electrophoresis followed by the Qiagen gel extractionprotocol. The Mariner Erm Gene Kelly was removed from pHGK5erm3 bydigestion with the restriction endonucleases BglII and SmaI (NEB), andpurified by agarose gel electrophoresis followed by the Qiagen gelextraction protocol. The products were ligated (according to Gibcoprotocol for T4 DNA ligase) overnight at 16° C. The ligation mixture wasthen transformed into E.coli PIR1 cells (Epicentre). The transformedcells were then plated on L-Agar (Sigma) plates containing Kanamycin at30 μg/ml and incubated overnight at 37° C. 12 colonies were then pickedand grown up overnight in LB-Kan 30 μg/ml and 1.5 ml of culture was usedfor DNA-mini-preps according to the Qiagen protocol. Restrictiondigestion with EcoRI and XbaI (NEB), followed by agarose gelelectrophoresis indicated that one clone contained the correct plasmid(pMARGK2b). DNA sequencing of pMARGK2b was performed on a Beckman CEQDNA sequencer, (using manufacturers recommended conditions) with thesequencing primers:

-   -   12 5′-AAG ATA CTG CAC TAT CAA CAC ACT C-3′ (SEQ ID NO: 16)    -   107 5′-CTACCCTGTGGAACACCTACATCT-3′ (SEQ ID NO: 4),        and found to contain the desired DNA sequence. This plasmid also        contains a temperature sensitive origin for replication in S.        aureus at temperatures of 30° C. or below, plus a        chloramphenicol resistance marker providing resistance in S.        aureus at 5 μg/ml.

pMARGK2b was introduced into S. aureus RN4220 by electroporation (0.5 μgpMARGK2b: 2.3 kV (0.1 cm cuvette), 25 μF, 100Ω) using a Gene Pulser(Biorad). The electroporation was plated out onto BHI-agar (Oxoid)containing chloramphenicol and erythromycin at 5 μg/ml. 6 colonies werethen picked and grown up overnight at 30° C. in BHI containingerythromycin and chloramphenicol at 5 μg/ml and 1.5 ml of culture wasused for plasmid DNA-mini-preps according to the Qiagen protocol.Plasmid DNA was restriction digested with EcoRI and XbaI and analysed byagarose gel electrophoresis. The restriction pattern of all 6 clonesmatched that obtained following the same digestion of pMARGK2b isolatedfrom E. coli.

Transducing bacteriophage φ11 was propagated (Novick, R. P. 1991.Genetic systems in staphylococci. Methods Enzymol. 204:587-636) from S.aureus RN4220 pMARGK1-6 and used to transduce the 6 plasmids intovirulent S. aureus SH1000, generating S. aureus SH1000 pMARGK1-6. 2colonies were then picked from each transduction and grown up overnightat 30° C. in BHI containing erythromycin and chloramphenicol at 5 μg/mland 1.5 ml of culture was used for plasmid DNA-mini-preps according tothe Qiagen protocol. Plasmid DNA was restriction digested with EcoRI andXbaI and analysed by agarose gel electrophoresis. The restrictionpattern of clones 1-5 matched that obtained following the same digestionof pMARGK2b isolated from E. coli. S. aureus SH1000 pMARGK3a was chosenas the parent strain for the generation of a transposon library.

Example 12 Generation of S. aureus Strain SH1000 Containing Mariner ErmGene Kelly, and the Mariner Transposase Gene

Plasmid pSPT246, containing the Mariner transposase and a tetracyclineresistance gene, was introduced into S. aureus SH1000 pMARGK3a bytransducing the plasmid from S. aureus SH1000 pSPI246 (isolate 3a) usingφ11-transducing bacteriophage. Following transduction, bacteria werepropagated on agar containing chloramphenicol, erythromycin andtetracycline all at 5 μg/ml at 30° C. for 48 h. Transduction yieldedapproximately 2000 colonies. All colonies were extracted from the topagar and inoculated into 600 ml BHI broth containing chloramphenicol,erythromycin and tetracycline all at 5 μg/ml and incubated o/n at 30° C.Bacteria (100 ml) were centrifuged at 4000×g for 5 min and resuspendedin 5 ml BHI broth containing 50% (v/v) glycerol and stored in 0.5 mlaliquots at −80° C.

Example 13 Generation of a Mariner Erm Gene Kelly Transposon Library inS. aureus SH1000

S. aureus SH1000 pMARGK3a pSRT146-3a (0.5 ml glycerol stock from above)was inoculated into 100 ml of room temperature BHI broth containingchloramphenicol, erythromycin and tetracyclin all at 5 μg/ml andincubated at 37° C. until the culture reached an A₆₀₀ of 0.4. 30 ml ofthis culture was centrifuged at 4000×g for 5 min and the pelletresuspended in 600 ml BHI broth containing 5 μg/ml erythromycin at 44°C. This culture was incubated at 44° C. until the culture reached anA₆₀₀ of 0.4. 30 ml of this culture was centrifuged at 4000×g for 5 minand the pellet resuspended in 600 ml BHI broth containing 5 μg/mlerythromycin at 44° C. This culture was incubated at 44° C. overnight.The resulting bacteria were tested and confirmed as sensitive totetracyclin and chloramphenicol whilst maintaining resistance toerythromycin. This indicated that transposition of Mariner Erm GeneKelly into the S. aureus SH1000 chromosome had occurred. Chromosomal DNAwas prepared from 200 ml of this culture for subsequent TMDH protocols.Glycerol stocks of this culture were prepared by centrifugation of 100ml of this culture at 4000×g for 5 min and resuspending the bacterialpellet in BHI containing 50% glycerol. Chromosomal DNA was prepared from112 colonies generated from the library generation protocol andsequenced using primer 199 5′ TAGCCAGTTTCGTCGTTAAATGCCC 3′ (SEQ ID NO:18) that binds 310 bases 5′ of the end of the transposon. InterpretableDNA sequence from 86 strains where the DNA flanking the end of thetransposon matched S. aureus DNA sequences located in the publicdatabases, indicating transposition of the Mariner Erm Gene Kellytransposon into the S. aureus SH1000 chromosome. A comparison of thelocation of these transposon insertions relative to the completechromosomal DNA sequence of S. aureus strain MW2 is shown in FIG. 8.This reveals the random nature of Mariner transposition into the S.aureus chromosome. This is compared to 59 S. aureus SH1000 Tn917mutants, and 50 S. aureus SH1000 Tn551 mutants made using thetransposons Tn917 and Tn551, respectively. Analysis of this data revealsthat both Tn551 and Tn917 have a transposition hotspot encompassing aregion of approximately 60 kb where about half of the mutants derivedfrom each transposon were located. Mutants generated using Mariner ErmGene Kelly do not appear to have such a sequence preference andtherefore libraries generated using this system will be of a much higherquality than those generated using either Tn917 or Tn551. In essenceMariner is much more suited to the generation of libraries suited toTMDH analysis of the S. aureus genome.

Example 14 Evaluation of the Mariner Erm Gene Kelly for TMDH

(i) T7 and SP6 RNA Polymerase Evaluation

In order to evaluate the novel transposon as suitable for the TMDHprotocol, iPCR reactions amplifying both ends of the transposon as wellas the DNA flanking the site of transposition from one of the sequencedtransposon mutants were used as template in an in vitro transcription(IVT) reactions using both SP6 (SP6 Megascript kit; Ambion) and T7 (T7Megashortscipt kit, Ambion) RNA polymerases, according to Ambionprotocols. RNA produced was purified using the Qiagen RNeasy mini Kit,and the amount of RNA produced measured at A₂₆₀. Transcription wasobserved both with the SP6 and T7 IVT reactions, but only when thespecific RNA polymerase was included in a reaction containing the iPCRproduct bearing the cognate RNA polymerase binding site. This indicatesno transcription occurs as a consequence of exposure of the T7 and SP6RNA polymerases, respectively, to the SP6 and T7 RNA polymerase promotersequences.

1-47. (canceled)
 48. A transposon which comprises an RNA polymeraserecognition site and a homing endonuclease recognition site.
 49. Atransposon according to claim 48 which comprises two RNA polymeraserecognition sites.
 50. A transposon according to claim 49, wherein thetwo RNA polymerase recognition sites are diverse.
 51. A transposonaccording to claim 50, wherein the two diverse RNA polymeraserecognition sites are two of a T7 RNA polymerase recognition site, anSP6 RNA polymerase recognition site or a T3 RNA polymerase recognitionsite.
 52. A transposon according to claim 48 which comprises two homingendonuclease recognition sites.
 53. A transposon according to claim 52,wherein the two homing endonuclease recognition sites are diverse.
 54. Atransposon according to claim 53, wherein the two diverse homingendonuclease recognition sites are an I-SceI recognition site and aPI-PspI recognition site.
 55. A transposon according to claim 48 whichfurther comprises a bacterial origin of replication.
 56. A transposonaccording to claim 48 which is a modified Tn5 transposon or a modifiedMariner transposon.
 57. A method for identifying an essential gene of anorganism, which method comprises: (i) providing a library of transposoninsertion mutants of the said organism, wherein the transposon is atransposon according to claim 48; (ii) isolating chromosomal DNA fromthe library of (i); (iii) digesting the chromosomal DNA with arestriction endonuclease that is capable of cutting 5′ to the RNApolymerase recognition site(s) in the transposon and 3′ to the RNApolymerase recognition site(s) in the chromosomal DNA flanking thetransposon insertion site; (iv) transcribing the resulting digested DNAfrom the RNA polymerase recognition site(s) in the said DNA; (v)hybridizing the resulting RNA with an oligonucleotide array; and (vi)identifying at least one probe on the oligonucleotide array whichcorresponds to an essential gene of the organism.
 58. A method accordingto claim 57, wherein a labeled ribonucleotide is present whentranscribing the digested DNA in step (iv).
 59. A method according toclaim 58, wherein step (v) is replaced by: (v)′ reverse transcribing theresulting RNA; and (v)″ hybridizing the resulting cDNA with anoligonucleotide array.
 60. A method according to claim 59, wherein alabeled deoxyribonucleotide is present when reverse transcribing the RNAin step (v)′.
 61. A method according to claim 57, wherein: (a) thetransposon comprises two RNA polymerase recognition sites which arediverse; (b) step (iv) is carried out by transcribing one aliquot of thedigested DNA with a first RNA polymerase and transcribing a secondaliquot of the digested DNA with a second different RNA polymerase; and(c) step (v) is carried out by hybridizing the two resulting RNA poolswith the same oligonucleotide array or separately with two copies of thesame oligonucleotide array.
 62. A method according to claim 61, whereinin step (b) the two aliquots of digested DNA are each transcribed in thepresence of a different labeled ribonucleotide.
 63. A method accordingto claim 59, wherein: (a) the transposon comprises two RNA polymeraserecognition sites which are diverse; (b) step (iv) is carried out bytranscribing one aliquot of the digested DNA with a first RNA polymeraseand transcribing a second aliquot of the digested DNA with a seconddifferent RNA polymerase; and (c) step (v)″ is carried out byhybridizing the two resulting cDNA pools with the same oligonucleotidearray or separately with two copies of the same oligonucleotide array.64. A method according to claim 63, wherein the two aliquots of RNAresulting from step (b) are each reverse transcribed using a differentlabeled deoxyribonucleotide.
 65. A method according to claim 57,wherein: (a) aliquots of the chromosomal DNA are digested separatelywith different restriction endonucleases in step (iii); (b) each of therestriction endonucleases is capable of cutting 5′ to the RNA polymeraserecognition site(s) in the transposon and 3′ to the RNA polymeraserecognition site(s) in the chromosomal DNA flanking the transposoninsertion site; and (c) each aliquot is subsequently treated separatelyin steps (iv) to (vi).
 66. A method according to claim 65, wherein twoor three aliquots of the chromosomal DNA are each digested separatelywith different restriction endonucleases.
 67. A method according toclaim 57, wherein step (iii) is replaced by: (iii)′ digesting thechromosomal DNA with a homing endonuclease which is capable of cutting5′ to RNA polymerase recognition site(s) in the transposon; (iii)″digesting the chromosomal DNA with a restriction endonuclease that iscapable of cutting 3′ to the RNA polymerase recognition site(s) in thechromosomal DNA flanking the transposon insertion site; and (iii)′″ligating the digested DNA with a biotinylated linker; and (iii)″″recovering the digested DNA using streptavidin-coated particles.
 68. Amethod for identifying a conditional essential gene of an organism,which method comprises: (a) providing a first sample of a library oftransposon insertion mutants of the said organism (input library); (b)providing a second sample of the library and subjecting that sample to aconditional restraint; (c) collecting the mutants that survive theconditional restraint in step (ii) to give a second library (outputlibrary); (d) carrying out a method according to steps (ii) to (iv) ofclaim 57 on the input library from step (a) and on the output libraryfrom step (c); (e) hybridizing the transcribed RNA derived from theinput library and from the output library separately to copies of thesame oligonucleotide array or, if the RNA derived from the two librariesis differentially labeled, to the same oligonucleotide array; and (f)identifying at least one probe on the oligonucleotide array(s) whichcorresponds to a conditional essential gene of the organism.
 69. Amethod for identifying a conditional essential gene of an organism,which method comprises: (a) providing a first sample of a library oftransposon insertion mutants of the said organism (input library); (b)providing a second sample of the library and subjecting that sample to aconditional restraint; (c) collecting the mutants that survive theconditional restraint in step (ii) to give a second library (outputlibrary); (d) carrying out a method according to steps (ii) to (v)′ ofclaim 59 on the input library from step (a) and on the output libraryfrom step (c); (e) hybridizing the reverse transcribed cDNA derived fromthe input library and from the output library separately to copies ofthe same oligonucleotide array or, if the cDNA derived from the twolibraries is differentially labeled, to the same oligonucleotide array;and (f) identifying at least one probe on the oligonucleotide array(s)which corresponds to a conditional essential gene of the organism.
 70. Amethod according to claim 68, wherein the organism is a bacterium andthe conditional restraint is growth of that bacterium in its host.
 71. Amethod according to claim 57 or 68, wherein the oligonucleotide arraycomprises probes which are from 9 to 150 bp in length and/or comprises 1probe for every 60 to 250 bp of the locus or loci represented on thearray.
 72. A method for identifying an inhibitor of transcription and/ortranslation of an essential or conditional essential gene and/or aninhibitor of activity of a polypeptide encoded by a said gene, whichmethod comprises: (a) identifying an essential or conditional essentialgene by a method according to claim 57 or 68; and (b) determiningwhether a test substance can inhibit transcription and/or translation ofa gene identified in step (a) and/or activity of a polypeptide encodedby a said identified gene, thereby to identify a said inhibitor.
 73. Aninhibitor identified by a method according to claim
 72. 74. An inhibitoraccording to claim 73 which is an antibody.
 75. An inhibitor accordingto claim 74 which is a monoclonal antibody.
 76. A pharmaceuticalcomposition comprising an inhibitor according to claim 73 wherein theessential or conditional essential gene is a bacterial, fungal oreukaryotic parasite essential or conditional essential gene and apharmaceutically acceptable carrier or diluent.
 77. A method of treatinga host suffering from a bacterial, fungal or eukaryotic parasiteinfection, which method comprises the step of administering to the hosta therapeutically effective amount of an inhibitor according to claim 73wherein the essential or conditional essential gene is a bacterial,fungal or eukaryotic parasite essential or conditional essential gene.78. A method for the preparation of a pharmaceutical composition, whichmethod comprises: (a) identifying an inhibitor of transcription and/ortranslation of an essential or conditional essential gene of an organismand/or an inhibitor of activity of a polypeptide encoded by a said geneby a method according to claim 72 wherein the essential or conditionalessential gene is a bacterial, fungal or eukaryotic parasite essentialor conditional essential gene; and (b) formulating the inhibitor thusidentified with a pharmaceutically acceptable carrier or diluent.
 79. Amethod for treating a host suffering from a bacterial, fungal oreukaryotic parasite infection, which method comprises: (a) identifyingan inhibitor of transcription and/or translation of an essential orconditional essential gene of an organism and/or an inhibitor ofactivity of a polypeptide encoded by a said gene by a method accordingto claim 72 wherein the essential or conditional essential gene is abacterial, fungal or eukaryotic parasite essential or conditionalessential gene; (b) formulating the inhibitor thus identified with apharmaceutically acceptable carrier or diluent; and (c) administering tothe host a therapeutically effective amount of an inhibitor thusformulated.
 80. An inhibitor according to claim 73, wherein theessential or conditional essential gene is a plant bacterial, plantfungal, plant pest or plant essential or conditional essential gene. 81.A bacterium attenuated by a non-reverting mutation in one or more genesidentified by a method according to claim
 68. 82. A vaccine comprising abacterium according to claim 81 and a pharmaceutically acceptablecarrier or diluent.
 83. A vaccine comprising a peptide encoded by anessential or conditional essential gene sequence identified by a methodaccording to claim 57 or
 68. 84. A method for raising an immune responsein a mammalian host, which method comprises the step of administering tothe host a bacterium according to claim 81 or a peptide encoded by anessential or conditional essential gene sequence identified by a methodaccording to claim 57 or
 68. 85. A method for preparing an attenuatedbacterium, which method comprises: (a) identifying a conditionalessential gene in a bacterium by a method according to claim 68; and (b)introducing a non-reverting mutation into a thus-identified conditionalessential gene of the bacterium, thereby to attenuate the bacterium. 86.A method for the preparation of a vaccine, which method comprises: (a)identifying a conditional essential gene in a bacterium by a methodaccording to claim 68; (b) introducing a non-reverting mutation into athus-identified conditional essential gene of the bacterium, thereby toattenuate the bacterium; and (c) formulating the attenuated bacteriumwith a pharmaceutically acceptable carrier or diluent.
 87. A method forthe preparation of a vaccine, which method comprises: (a) identifying anessential or conditional essential gene sequence by a method accordingto claim 57 or 68; and (b) formulating a peptide encoded by athus-identified essential or conditional essential gene sequence with apharmaceutically acceptable carrier or diluent.
 88. A method for raisingan immune response in a mammalian host, which method comprises: (a)identifying a conditional essential gene in a bacterium by a methodaccording to claim 68; (b) introducing a non-reverting mutation into athus-identified conditional essential gene of the bacterium, thereby toattenuate the bacterium; (c) formulating the attenuated bacterium with apharmaceutically acceptable carrier or diluent; and administering to thehost the attenuated bacterium thus formulated.
 89. A method for raisingan immune response in a mammalian host, which method comprises: (a)identifying an essential or conditional essential gene sequence by amethod according to claim 57 or 68; (b) formulating a peptide encoded bya thus-identified essential or conditional essential gene sequence witha pharmaceutically acceptable carrier or diluent; and (c) administeringto the host the peptide thus formulated.